Methods and compositions for detecting a drug resistant EGFR mutant

ABSTRACT

Development of acquired resistance to the therapeutic effects of an epidermal growth factor (EGFR) tyrosine kinase inhibitor in a patient that is suffering from a cancer is predicted by:
         (a) obtaining a sample from the patient, wherein the cancer harbors a somatic gain-of-function mutation in the tyrosine kinase domain of EGFR that enhances the sensitivity of the cancer to the tyrosine kinase inhibitor, and   (b) testing the sample to determine whether the gene encoding EGFR is present in a mutant form that encodes a T790M mutant of EGFR in addition to the somatic gain of function mutation. A finding that the mutant form is present indicates that the cancer has become or will become resistant to the EGFR tyrosine kinase inhibitor.

FIELD OF THE INVENTION

This invention relates to a method for testing for a mutation in theepidermal growth factor receptor (EGFR) gene or EGFR protein, suchmutation being the underlying reason for resistance to certain cancertherapies directed towards inhibiting EGFR. This invention furtherrelates to methods for developing new therapies that inhibit said mutantEGFR.

BACKGROUND OF THE INVENTION

The epidermal growth factor receptor (EGFR) has been identified as arelevant target for treatment of solid tumors, as it is involved inregulating cellular functions important in the proliferation andsurvival of cancer cells. EGFR is commonly expressed in a range oftumors, and high expression is often related to poor prognosis. A newclass of targeted therapies directed at inhibiting the EGFR, tyrosinekinase inhibitors, have appeared. Two known examples are gefitinib(Iressa) or erlotinib (Tarceva). Despite initial responses of somepatients to these therapies, patients eventually progress by unknownmechanisms of “acquired” resistance.

EGFR has been thought to play an important role in lung cancer. Howeveronly a small portion non-small cell lung cancers (NSCLCs) respond toIressa or Tarceva (see FIG. 1 for structures). Lung adenocarcinomas frompatients who respond to the tyrosine kinase inhibitors gefitinib orerlotinib usually harbor somatic gain-of-function mutations in exonsencoding the tyrosine kinase domain of EGFR. Such mutations are found inabout 10% of NSCLCs from the United States [1,2,3], with higherincidences observed in east Asia [2,4,5,6]. Some 90% of NSCLC-associatedmutations occur as either multi-nucleotide in-frame deletions in exon19, involving elimination of four amino acids, Leu-Arg-Glu-Ala, or as asingle nucleotide substitution at nucleotide 2573 (T→G) in exon 21,resulting in substitution of arginine for leucine at position 858(L858R). Both of these mutations are associated with sensitivity to thesmall-molecule kinase inhibitors gefitinib or erlotinib[1,2,3].Unfortunately, nearly all patients who experience marked improvement onthese drugs eventually develop progression of disease. While KRAS(v-Ki-ras2, Kirsten rat sarcoma viral oncogene homolog, a RAS familymember) mutations have been associated with some cases of primaryresistance to gefitinib or erlotinib [7], mechanisms underlying“acquired” or “secondary” resistance are unknown.

Therefore there is a need in the art for the determining the underlyingcauses of such resistance so that a diagnostic test can be developed anda more effective treatment provided. Moreover, there is a need in theart for new compounds that are able to treat patients that show cancerprogression or relapse despite initial response to current EGFRinhibitors.

SUMMARY OF THE INVENTION

The present invention provides polymerase chain reaction primersdirected to detecting the EGFR mutant C→T at the position correspondingto base 2369 of EGFR cDNA. This mutation encodes a change in the EGFRprotein from threonine in the wild type to methionine in the mutant atposition 790. This mutation is shown to be sparse in patients before orin early stages of treatment with gefitinib or erlotinib. But since themutation abrogates sensitivity to these agents, cancer cells harboringthe mutation are positively selected, leading to patients that arerefractory to further treatment. The invention further provides methodsto detect the mutation in patients, whose ultimate objective is earlyidentification of refractory cases so that alternative treatments can beinitiated.

In a first aspect the invention provides a PCR primer that hybridizesunder suitable PCR conditions to a sense strand or to an antisensestrand of a polynucleotide sequence 5′ in each respective strand to amutation of an EGFR gene that encodes a substitution of threonine bymethionine at position 790 of the EGFR polypeptide, wherein the PCRprimer binds within 200 nucleotides of said mutation. General primerstructures are provided based on SEQ ID NOS:4-7 and 12-15 that may belarger or smaller than these particular sequences, as well as primerswhose sequences may have a certain number of bases in the sequencesgiven by SEQ ID NOS:4-7 and 12-15 that are substituted by other bases.

In another aspect the invention provides a PCR primer that hybridizesunder suitable PCR conditions to a first polynucleotide encoding a wildtype EGFR polypeptide, or a polynucleotide fragment thereof, wherein theprimer hybridizes to the sense strand sequence or to the antisensestrand sequence that includes the wild type C at the positioncorresponding to base 2369 of EGFR cDNA and wherein the primerhybridizes weakly or not at all to a second EGFR polynucleotidecontaining a mutant T at position 2369 under the PCR conditions.

In still a further embodiment the invention provides a PCR primer thathybridizes under suitable PCR conditions to a first polynucleotideencoding a mutant EGFR polypeptide, or a polynucleotide fragmentthereof, wherein the primer hybridizes to the sense strand sequence orto the antisense strand sequence that includes a mutant T at theposition corresponding to base 2369 of EGFR cDNA and wherein the primerhybridizes weakly or not at all to a second EGFR polynucleotidecontaining a wild type C at position 2369 under the PCR conditions.General primer structures are provided based on SEQ ID NOS:12 and 13that may be larger or smaller than these particular sequences, as wellas primers whose sequences may have a certain number of bases in thesequences given by SEQ ID NOS:12 and 13 that are substituted by otherbases.

In still an additional aspect the invention provides a method ofdetecting a mutant epidermal growth factor receptor (EGFR) gene in asample that includes probing the sample with a means for selectivelydetecting a nucleotide sequence containing a mutant T at the positioncorresponding to base 2369 of EGFR cDNA, and identifying that the baseat said position is T. In a significant embodiment the meansdistinguishes between detecting a mutant T and a wild type C at saidposition.

In common embodiments the sample includes tissue or cells that are orare suspected of being cancerous or malignant. Such samples originate ina subject having or suspected of having a cancer or malignant tumor, andmay be obtained by biopsy or similar surgical procedures.

In certain prevalent embodiments of this method the probing includessteps of

-   -   a) if necessary, treating the sample to liberate the nucleic        acids contained therein;    -   b) contacting the nucleic acids obtained from the sample with a        composition that includes a first PCR primer that hybridizes to        the sense strand sequence or to the antisense strand sequence        that includes the mutant T at the position corresponding to base        2369 of EGFR cDNA and wherein the primer hybridizes weakly or        not at all to a second EGFR polynucleotide containing a wild        type C at position 2369 under the PCR conditions; and    -   c) carrying out a PCR reaction in the presence of a second PCR        primer to provide a PCR amplicon containing a mutant T at the        position corresponding to base 2369.        The PCR reaction may advantageously incorporate a label into the        PCR amplicon; this permits the identifying step to include        detecting the label.

In alternative frequent embodiments of this method the probing includessteps of

-   -   a) if necessary, treating the sample to liberate the nucleic        acids contained therein;    -   b) contacting the nucleic acids obtained from the sample with a        composition that includes a pair of polymerase chain reaction        (PCR) primers that hybridize under suitable PCR conditions to a        polynucleotide encoding an EGFR polypeptide wherein the pair of        primers brackets the position corresponding to base 2369 of EGFR        cDNA to provide a PCR mixture;    -   c) carrying out a PCR reaction on the mixture to provide a PCR        amplicon containing the position corresponding to base 2369; and    -   d) contacting the amplicon with a cleaving means that cleaves        the amplicon either        -   i) by cleaving an amplicon having a mutant T at the position            corresponding to base 2369 within 6 bases of the position            but not so cleaving an amplicon having a wild type C at the            position, or        -   ii) by cleaving an amplicon having a wild type C at the            position corresponding to base 2369 within 6 bases of the            position but not so cleaving an amplicon having a mutant T            at the position.            The PCR reaction may advantageously incorporate a label into            the PCR amplicon thus permitting the identifying to include            detecting a length polymorphisms of the cleaved labeled            polynucleotides.

In still further common embodiments of this method the probing includessteps of

-   -   a) if necessary, treating the sample to liberate the nucleic        acids contained therein;    -   b) immobilizing at least a portion of the nucleic acids obtained        from the sample on a solid support; and    -   c) contacting the immobilized nucleic acids with a probe        oligonucleotide that hybridizes to a polynucleotide encoding an        EGFR polypeptide wherein the sequence of the probe includes a        base complementary to a mutant T at the position corresponding        to base 2369 of EGFR cDNA and wherein the probe hybridizes        weakly or not at all to a polynucleotide containing a wild type        C at position 2369 under suitable hybridization conditions.

In a common embodiment of a method inverse to that just described theprobing includes steps of

-   -   a) if necessary, treating the sample to liberate the nucleic        acids contained therein;    -   b) immobilizing a probe oligonucleotide that hybridizes to a        polynucleotide encoding an EGFR polypeptide wherein the sequence        of the probe includes a base complementary to a mutant T at the        position corresponding to base 2369 of EGFR cDNA and wherein the        probe hybridizes weakly or not at all to a polynucleotide        containing a wild type C at position 2369 on a solid support;        and    -   c) contacting the immobilized probe with at least a portion of        the nucleic acids obtained from the sample under suitable        hybridization conditions.        In these embodiments involving a solid support the component        that binds to the immobilized partner includes a label and the        identifying includes detecting the label.

In yet another aspect the invention provides a method of predictingresistance to the therapeutic effects of gefitinib or erlotinib in asubject suffering from or suspected of having a cancer. This methodemploys the steps described in the method of detecting a mutantepidermal growth factor receptor (EGFR) gene in a sample described inthe preceding paragraphs, and concludes that upon a positive finding ofa mutant at position 2369 the subject is predicted to be resistant totreatment by gefitinib or erlotinib.

In a further aspect the invention provides a kit that includes at leastone container and, contained therein, a composition that includes atleast one PCR primer described in the preceding paragraphs. In certainembodiments the kit further includes a cleaving means that cleaves anEGFR polynucleotide either

-   -   a) by cleaving a polynucleotide having a mutant T at the        position corresponding to base 2369 of EGFR cDNA within 6 bases        of the position but not so cleaving a polynucleotide having a        wild type C at the position, or    -   b) by cleaving a polynucleotide having a wild type C at the        position corresponding to base 2369 of EGFR cDNA within 6 bases        of the position but not so cleaving a polynucleotide having a        mutant T at the position.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Chemical structures of gefitinib and erlotinib.

FIG. 2. Schematic representation of various embodiments of thepolynucleotides of the invention. The length is 200 nucleotides or less,and 11 nucleotides or greater. In c) the darker vertical barsdiagrammatically represent substituted nucleotides.

FIG. 3. A Novel PCR-RFLP Assay Independently Confimis Presence of theT790M Mutation in Exon 20 of the EGFR Kinase Domain

-   -   (A) Design of the assay (see text for details). “F” designates a        fluorescent label, such as FAM. At the bottom of this panel, the        assay demonstrates with the 97-bp NlaIII cleavage product the        presence of the T790M mutation in the H1975 cell line; this        product is absent in H2030 DNA. The 106-bp NlaIII cleavage        product is generated by digestion of wild-type EGFR.    -   (B) The PCR-RFLP assay demonstrates that pre-drug tumor samples        from the three patients lack detectable levels of the mutant        97-bp product, while specimens obtained after disease        progression contain the T790M mutation. Pt: patient.

FIG. 4. Imaging Studies from Patients 1, 2, and 3

-   -   (A) Patient 1. Serial chest radiographs from before (day 0) and        during gefitinib treatment (14 d and 9 mo), demonstrating        initial response and subsequent progression.    -   (B) Patient 2. Serial CT studies of the chest before (day 0) and        during erlotinib treatment (4 mo and 25 mo), demonstrating        initial response and subsequent progression.    -   (C) Patient 3. Serial chest radiographs before (day 0) and        during adjuvant gefitinib treatment (3 mo), following complete        resection of grossly visible disease. The left-sided pleural        effusion seen at 3 mo recurred 4 mo later, at which time fluid        was collected for molecular analysis.

FIG. 5. Re-Biopsy Studies The biopsy needles are indicated by whitearrows.

-   -   (A) Patient 1. CT-guided biopsy of progressing lung lesions        after 10 months on gefitinib (left panel). Two months later,        fluid from a right-sided pleural effusion (right panel) was        collected for molecular analysis.    -   (B) Patient 2. CT-guided biopsy of a progressing thoracic spine        lesion (left panel) and fluoroscopic-guided biopsy of a        progressing lung lesion (right panel).

FIG. 6. Sequencing Chromatograms with the EGFR Exon 19 and 21 MutationsIdentified in Patients 1 and 2

-   -   (A) Status of EGFR exon 21 in tumor specimens from patient 1.        DNA from the growing lung lesion and the pleural effusion        demonstrated a heterozygous T→G mutation at position 2573,        leading to the common L858R amino acid substitution.    -   (B) All three specimens from patient 2 showed the same        heterozygous exon 19 deletion, removing residues 747-749 and        changing the alanine at position 750 to proline. The partial        forward sequence shown for original lung tumor and growing lung        lesion is Seq ID No. 16. The partial forward sequence shown for        growing bine lesion is Seq ID No. 18. The partial reverse        sequences shown are all Seq ID No. 17. The original four-color        sequencing traces have been transformed to black-and-white.

FIG. 7. Sequencing Chromatograms with the T790M EGFR Exon 20 Mutation inVarious Clinical Specimens and the NSCLC Cell Line H1975. The originalfour-color sequencing traces have been transformed to black-and-white.

-   -   (A-C) In all three patients—patient 1 (A), patient 2 (B), and        patient 3 (C)—the secondary T790M mutation was observed only in        lesions obtained after progression on either gefitinib or        erlotinib. The partial forward sequence shown in FIG. 7A, for        growing lung lesion, and 7B are Seq ID No. 19. The partial        reserve sequence shown in FIG. 7A for growing lung lesion, and        7B are Seq ID No. 20. The partial forward sequence shown in FIG.        7A for pleural effusion is Seq ID No. 21. The partial reverse        sequence shown in FIG. 7A for pleural effusion is Seq ID No. 22.        The partial forward sequence shown in FIG. 7C for resected lung        tumor is Seq ID No. 19. The partial reverse sequence shown in        FIG. 7C for resected lung tumor is Seq ID No. 23. The partial        forward sequence shown in FIG. 7C for pleural effusion is Seq ID        No. 24. The partial reverse sequence shown in FIG. 7C for        pleural effusion is Seq ID No. 25.    -   (D) Cell line H1975 contains both an exon 21 L858R mutation        (upper panel) and the exon 20 T790M mutation (lower panel). The        asterisks indicate a common SNP (A or G) at nucleotide 2361; the        arrows indicate the mutation at nucleotide 2369 (C→T), which        leads to substitution of methionine (ATG) for threonine (ACG) at        position 790. In the forward direction, the mutant T peak is        blue. In the reverse direction, the mutant peak is green, while        the underlying blue peak represents an “echo” from the adjacent        nucleotide. The partial forward sequence shown in FIG. 7D for        exon is Seq ID No. 26. The partial reverse sequence shown in        FIG. 7D for exon 20 is Seq ID No. 27.

FIG. 8. EGFR Mutants Containing the T790M Mutation Are Resistant toInhibition by Gefitinib or Erlotinib

293T cells were transiently transfected with plasmids encoding wild-type(WT) EGFR or EGFR mutants with the following changes: T790M, L858R,L858R+T790M, del L747−E749;A750P, or del L747+E749;A750P+T790M. After 36h, cells were serum-starved for 24 h, treated with gefitinib orerlotinib for 1 h, and then harvested for immunoblot analysis usinganti-p-EGFR (Y1092), anti-t-EGFR, anti-phosphotyrosine (p-Tyr), andanti-actin antibodies. The EGFR T790M mutation, in conjunction witheither wild-type EGFR or the drug-sensitive L858R EGFR mutant, preventsinhibition of tyrosine phosphorylation (A) or p-EGFR (B) by gefitinib.Analogously, the T790M mutation, in conjunction with the drug-responsivedel L747−E749;A750P EGFR mutant, prevents inhibition of p-EGFR byerlotinib (C).

FIG. 9. Sensitivity to Gefitinib Differs Among NSCLC Cell LinesContaining Various Mutations in EGFR or KRAS

The three indicated NSCLC cell lines (H3255: L858R mutation; H1975: bothT790M and L858R mutations; and H2030: wild-type EGFR, mutant KRAS (seeTable 7)) were grown in increasing concentrations of gefitinib, and thedensity of live cells after 48 hours of treatment was measured using aCalcein AM fluorescence assay. Fluorescence in vehicle-treated cells isexpressed as 100%. Results are the mean±SE of three independentexperiments in which there were four to eight replicates of eachcondition. Similar results were obtained with erlotinib.

FIG. 10. Sensitivity to Erlotinib Differs among NSCLC Cell LinesContaining Various Mutations in EGFR or KRAS. See legend for FIG. 9.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent application publications, and patent applicationsidentified herein are incorporated by reference in their entireties, asif appearing herein verbatim. All technical publications identifiedherein are also incorporated by reference.

Abbreviations: CML, chronic myelogenous leukemia; CT, computedtomography; del, deletion; EGFR, epidermal growth factor receptor; GIST,gastrointestinal stromal tumor; HES, hypereosinophilic syndrome; NSCLC,non-small cell lung cancer; p-EGFR, phospho-EGFR; PCR-RFLP, PCRrestriction fragment length polymorphism; SNP, single nucleotidepolymorphism; t-EGFR, total EGFR

Accession Numbers: Reference EGFR sequence was obtained from LocusLinkaccession number 1956 and GenBank accession number NT 033968.

Two numbering systems are used for EGFR. The first denotes theinitiating methionine in the signal sequence as amino acid −24. Thesecond, used here, denotes the methionine as amino acid +1. Commercialsuppliers of antibodies, such as the Y1068-specific anti-phospho-EGFR,use the first nomenclature. To be consistent, we consider Y1068 asY1092. Likewise, the T790M mutation reported here has also been calledT766M.

In the present description, the articles “a”, “an”, and “the” relateequivalently to a meaning as singular or as plural. The particular sensefor these articles is apparent from the context in which they are used.

As used herein the term “tumor” refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all precancerous andcancerous cells and tissues.

As used herein the term “cancer” refers to cells or tissues possessingcharacteristics such as uncontrolled proliferation, loss of specializedfunctions, immortality, significant metastatic potential, significantincrease in anti-apoptotic activity, rapid growth and proliferationrate, and certain characteristic morphological and cellular markers. Insome circumstances, cancer cells will be in the form of a tumor; suchcells may exist locally within an animal, and in other circumstancesthey may circulate in the blood stream as independent cells, forexample, leukemic cells.

To determine whether cancers that acquire clinical resistance to eithergefitinib or erlotinib display additional mutations in the EGFR kinasedomain, we have examined the status of EGFR exons 18 to 24 in tumorsfrom thirteen patients who initially responded but subsequentlyprogressed while on these drugs. These exons were also assessed in tumorcells from a fourteenth patient whose disease rapidly recurred while ongefitinib therapy after complete gross tumor resection. Because of theassociation of KRAS mutations with primary resistance to gefitinib anderlotinib [7], we also examined the status of KRAS in tumor cells fromthese six patients. In an effort to explain the selective advantage ofcells with a newly identified “resistance” mutation in EGFR—a T790Mamino acid substitution (also known as T766M), a 2369 C→T change in theEGFR genomic sequence—we further characterized the drug sensitivity ofputatively resistant EGFR mutants versus wild-type or drug-sensitiveEGFR mutants, using both a NSCLC cell line fortuitously found to containthe T790M mutation and lysates from cells transiently transfected withwild-type and mutant EGFR cDNAs.

SEQ ID NO:1 (shown in Table 1) displays the cDNA sequence of the mutanthuman EGFR gene. The pair of primers used to amplify the EGFR fragmentused for sequencing to detect the presence or absence of the EGFR T790Mmutation is underlined and in italic font. The mutant t2369 nucleotideis shown in enlarged bold font. The wild type EGFR sequence is knownfrom GenBank Accession No. X00588, and Ullrich, A. et al. “Humanepidermal growth factor receptor cDNA sequence and aberrant expressionof the amplified gene in A431 epidermoid carcinoma cells”, Nature 309(5967), 418-425 (1984). The translated mutant protein sequence is shownin SEQ ID NO:2 (Table 2). The mutant M790 is shown in enlarged boldfont.

TABLE 1 2369 C→T MUTANT EGFR cDNA (SEQ ID NO: 1)atgcgaccctccgggacggccggggcagcgctcctggcgctgctggctgcgctctgcccggcgagtcgggctctggaggaaaagaaagtttgccaaggcacgagtaacaagctcacgcagttgggcacttttgaagatcattttctcagcctccagaggatgttcaataactgtgaggtggtccttgggaatttggaaattacctatgtgcagaggaattatgatctttccttcttaaagaccatccaggaggtggctggttatgtcctcattgccctcaacacagtggagcgaattcctttggaaaacctgcagatcatcagaggaaatatgtactacgaaaattcctatgccttagcagtcttatctaactatgatgcaaataaaaccggactgaaggagctgcccatgagaaatttacaggaaatcctgcatggcgccgtgcggttcagcaacaaccctgccctgtgcaacgtggagagcatccagtggcgggacatagtcagcagtgactttctcagcaacatgtcgatggacttccagaaccacctgggcagctgccaaaagtgtgatccaagctgtcccaatgggagctgctggggtgcaggagaggagaactgccagaaactgaccaaaatcatctgtgcccagcagtgctccgggcgctgccgtggcaagtcccccagtgactgctgccacaaccagtgtgctgcaggctgcacaggcccccgggagagcgactgcctggtctgccgcaaattccgagacgaagccacgtgcaaggacacctgccccccactcatgctctacaaccccaccacgtaccagatggatgtgaaccccgagggcaaatacagctttggtgccacctgcgtgaagaagtgtccccgtaattatgtggtgacagatcacggctcgtgcgtccgagcctgtggggccgacagctatgagatggaggaagacggcgtccgcaagtgtaagaagtgcgaagggccttgccgcaaagtgtgtaacggaataggtattggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcctgccggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatcacagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggaccaagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggagatgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaattataagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggcccggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgagccaagggagtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacggggaccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaaacaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggccaggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtggccctggggatcggcctcttcatgcgaaggcgccacatcgttcggaagcgcacgctgcggaggctgctgcaggagagggagcttgtggagcctcttacacccagtggagaagct cccaaccaagctctcttgag gatcttgaaggaaactgaattcaaaaagatcaaagtgctgggctccggtgcgttcggcacggtgtataagggactctggatcccagaaggtgagaaagttaaaattcccgtcgctatcaaggaattaagagaagcaacatctccgaaagccaacaaggaaatcctcgatgaagcctacgtgatggccagcgtggacaacccccacgtgtgccgcctgctgggcatctgcctcacctccaccgtgcagctcatcatgcagctcatgcccttcggctgcctcctggactatgtccgggaacacaaagacaatattggctcccagtacctgctcaactggtgtgtgcagatcgcaaagggcatgaactacttggaggaccgtcgcttggtgcaccgcgacctggcagccaggaacgtactggtgaaaacaccgcagcatgtcaagatcacagattttgggctggccaaactgctgggtgcggaagagaaagaataccatgcagaaggaggcaaagtgcctatcaagtggatggcattggaatcaattttacacagaatctatacccaccagagtgatgtctggagctacggggtgaccgtttgggagttgatgacctttggatccaagccatatgacggaatccctgccagcgagatctcctccatcctggagaaaggagaacgcctccctcagccacccatatgtaccatcgatgtctacatgatcatggtcaagtgctggatgatagacgcagatagtcgcccaaagttccgtgagttgatcatcgaattctccaaaatggcccgaga cccccagcgctaccttgtcat tcagggggatgaaagaatgcatttgccaagtcctacagactccaacttctaccgtgccctgatggatgaagaagacatggacgacgtggtggatgccgacgagtacctcatcccacagcagggcttcttcagcagcccctccacgtcacggactcccctcctgagctctctgagtgcaaccagcaacaattccaccgtggcttgcattgatagaaatgggctgcaaagctgtcccatcaaggaagacagcttcttgcagcgatacagctcagaccccacaggcgccttgactgaggacagcatagacgacaccttcctcccagtgcctgaatacataaaccagtccgttcccaaaaggcccgctggctctgtgcagaatcctgtctatcacaatcagcctctgaaccccgcgcccagcagagacccacactaccaggacccccacagcactgcagtgggcaaccccgagtatctcaacactgtccagcccacctgtgtcaacagcacattcgacagccctgcccactgggcccagaaaggcagccaccaaattagcctggacaaccctgactaccagcaggacttctttcccaaggaagccaagccaaatggcatctttaagggctccacagctgaaaatgcagaatacctaagggtcgcgccacaaagcagtgaatttattggagcatga

TABLE 2 T790M MUTANT EGFR (SEQ ID NO: 2)MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCKLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQS

As used herein, a “nucleic acid” or “polynucleotide”, and similar termsand phrases, relate to polymers composed of naturally occurringnucleotides as well as to polymers composed of synthetic or modifiednucleotides. Thus, as used herein, a polynucleotide that is a RNA, or apolynucleotide that is a DNA, or a polynucleotide that contains bothdeoxyribonucleotides and ribonucleotides, may include naturallyoccurring moieties such as the naturally occurring bases and ribose ordeoxyribose rings, or they may be composed of synthetic or modifiedmoieties such as those described below. A polynucleotide employed in theinvention may be single stranded or it may be a base paired doublestranded structure, or even a triple stranded base paired structure.

Nucleic acids and polynucleotides may be 20 or more nucleotides inlength, or 30 or more nucleotides in length, or 50 or more nucleotidesin length, or 100 or more, or 1000 or more, or tens of thousands ormore, or hundreds of thousands or more, in length. As used herein,“oligonucleotides” and similar terms based on this relate to shortpolymers composed of naturally occurring nucleotides as well as topolymers composed of synthetic or modified nucleotides, as described inthe immediately preceding paragraph. Oligonucleotides may be 10 or morenucleotides in length, or 20 or more nucleotides in length, or 30 ormore nucleotides in length, or 40 or more, up to about 50, nucleotidesin length. Oligonucleotides may be chemically synthesized and may beused as PCR primers, or probes, among other uses.

It is understood that, because of the overlap in size ranges provided inthe preceding paragraph, the terms “polynucleotide” and“oligonucleotide” may be used synonymously herein to refer to primer ora probe of the invention.

As used herein “nucleotide sequence”, “oligonucleotide sequence” or“polynucleotide sequence”, and similar terms, relate interchangeablyboth to the sequence of bases that an oligonucleotide or polynucleotidehas, as well as to the oligonucleotide or polynucleotide structurepossessing the sequence. A nucleotide sequence or a polynucleotidesequence furthermore relates to any natural or synthetic polynucleotideor oligonucleotide in which the sequence of bases is defined bydescription or recitation of a particular sequence of lettersdesignating bases as conventionally employed in the field.

A “nucleoside” is conventionally understood by workers of skill infields such as biochemistry, molecular biology, genomics, and similarfields related to the field of the invention as comprising amonosaccharide linked in glycosidic linkage to a purine or pyrimidinebase; and a “nucleotide” comprises a nucleoside with at least onephosphate group appended, typically at a 3′ or a 5′ position (forpentoses) of the saccharide, but may be at other positions of thesaccharide. Nucleotide residues occupy sequential positions in anoligonucleotide or a polynucleotide. A modification or derivative of anucleotide may occur at any sequential position in an oligonucleotide ora polynucleotide. All modified or derivatized oligonucleotides andpolynucleotides are encompassed within the invention and fall within thescope of the claims. Modifications or derivatives can occur in thephosphate group, the monosaccharide or the base.

By way of nonlimiting examples, the following descriptions providecertain modified or derivatized nucleotides, all of which are within thescope of the polynucleotides of the invention. The monosaccharide may bemodified by being, for example, a pentose or a hexose other than aribose or a deoxyribose. The monosaccharide may also be modified bysubstituting hydryoxyl groups with hydro or amino groups, by alkylatingor esterifying additional hydroxyl groups, and so on. Substituents atthe 2′ position, such as 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-allyl, 2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group provide enhancedhybridization properties to an oligonucleotide.

The bases in oligonucleotides and polynucleotides may be “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Inaddition they may be bases with modifications or substitutions.Nonlimiting examples of modified bases include other synthetic andnatural bases such as hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil,5-halo-cytosine, 5-propy-uracil, 5-propynyl-cytosine and other alkynylderivatives of pyrimidine bases, 6-azo-uracil, 6-azo-cytosine,6-azo-thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino-,8-thiol-, 8-thioalkyl-, 8-hydroxyl- and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-fluoro-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified bases include tricyclic pyrimidinessuch as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases mayalso include those in which the purine or pyrimidine base is replacedwith other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine and 2-pyridone. Further bases include those disclosed inU.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, those disclosed by Englisch et al., AngewandteChemie, International Edition (1991) 30, 613, and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain ofthese bases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2.degree. C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Researchand Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications. See U.S. Pat. Nos.6,503,754 and 6,506,735 and references cited therein, incorporatedherein by reference. Modifications further include those disclosed inU.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugatedoligonucleotides; U.S. Pat. Nos. 5,212,295, 5,521,302, 5,587,361 and5,599,797, drawn to oligonucleotides incorporating chiral phosphoruslinkages including phosphorothioates; U.S. Pat. Nos. 5,378,825,5,541,307, and 5,386,023,drawn to oligonucleotides having modifiedbackbones; U.S. Pat. Nos. 5,457,191 and 5,459,255, drawn to modifiednucleobases; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids;U.S. Pat. No. 5,554,746, drawn to oligonucleotides having beta-lactambackbones; U.S. Pat. No. 5,571,902, disclosing the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, disclosing alkylthionucleosides; U.S. Pat. No. 5,506,351, drawn to 2′-O-alkyl guanosine,2,6-diaminopurine, and related compounds; U.S. Pat. No. 5,587,469, drawnto oligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, and U.S. Pat. No. 5,608,046, drawn to conjugated4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat.Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods ofsynthesizing 2′-fluoro-oligonucleotides.

The linkages between nucleotides is commonly the 3′-5′ phosphatelinkage, which may be a natural phosphodiester linkage, aphosphothioester linkage, and still other synthetic linkages.Oligonucleotides containing phosphorothioate backbones have enhancednuclease stability. Examples of modified backbones include,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphates.Additional linkages include phosphotriester, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridged phosphorothioateand sulfone internucleotide linkages. Other polymeric linkages include2′-5′ linked analogs of these. See U.S. Pat. Nos. 6,503,754 and6,506,735 and references cited therein, incorporated herein byreference.

Any modifications including those exemplified in the above descriptioncan readily be incorporated into, and are comprised within the scope of,the polynucleotides of the invention. Use of any modified nucleotide isequivalent to use of a naturally occurring nucleotide having the samebase-pairing properties, as understood by a worker of skill in the art.All equivalent modified nucleotides fall within the scope of the presentinvention as disclosed and claimed herein.

As used herein and in the claims, the term “complement”,“complementary”, “complementarity”, and similar words and phrases,relate to two sequences whose bases form complementary base pairs, baseby base, as conventionally understood by workers of skill in fields suchas biochemistry, molecular biology, genomics, and similar fields relatedto the field of the invention. Two single stranded (ss) polynucleotideshaving complementary sequences can hybridize with each other undersuitable buffer and temperature conditions to form a double stranded(ds) polynucleotide. By way of nonlimiting example, if the naturallyoccurring bases are considered, A and (T or U) interact with each other,and G and C interact with each other. Unless otherwise indicated,“complementary” is intended to signify “fully complementary”, namely,that when two polynucleotide strands are aligned with each other, therewill be at least a portion of the strands in which each base in asequence of contiguous bases in one strand is complementary to aninteracting base in a sequence of contiguous bases of the same length onthe opposing strand.

As used herein, “liberate” and similar words and phrases, when used inconnection with a nucleic acid, relate to a process whereby a cell or atissue is treated sufficiently to make the nucleic acids containedtherein available for interaction with reagents, including PCR primers,employed in methods of the present invention.

As used herein, “hybridize”, “hybridization” and similar words andphrases relate to a process of forming a nucleic acid, polynucleotide,or oligonucleotide duplex by causing strands with complementarysequences to interact with each other. The interaction occurs by virtueof complementary bases on each of the strands specifically interactingto form a pair. The ability of strands to hybridize to each otherdepends on a variety of conditions, as set forth below. Nucleic acidstrands hybridize with each other when a sufficient number ofcorresponding positions in each strand are occupied by nucleotides thatcan interact with each other. Polynucleotide strands that hybridize toeach other may be fully complementary. Alternatively, two hybridizedpolynucleotides may be “substantially complementary” to each other,indicating that they have a small number of mismatched bases. Bothnaturally occurring bases, and modified bases such as those describedherein, participate in forming complementary base pairs. It isunderstood by workers of skill in the field of the present invention,including by way of nonlimiting example biochemists and molecularbiologists, that the sequences of strands forming a duplex need not be100% complementary to each other to be specifically hybridizable.

As used herein “fragment” and similar words and phrases relate toportions of a nucleic acid, polynucleotide or oligonucleotide shorterthan the full sequence of a reference. The sequence of bases in afragment is unaltered from the sequence of the corresponding portion ofthe reference; there are no insertions or deletions in a fragment incomparison with the corresponding portion of the reference.

As used herein “cleaving means” and similar terms and phrases relate toa substance that cleaves a polynucleotide in a sequence-specificfashion. The cleaving means interacts only with a polynucleotide at asusceptible subsequence of bases present therein, and cleaves thepolynucleotide into two smaller pieces. Nonlimiting examples of cleavingmeans include restriction nucleases, sequence-specific ribozymes,aptamers with cleaving activity, and sequence-specific organic moleculeswith cleaving activity. Any equivalent cleaving means known to workersof skill in the field of the invention are within the scope of theinvention.

“Complementary DNA” (cDNA), is a single-stranded DNA molecule that iscopied from an mRNA template by the enzyme reverse transcriptase,resulting in a sequence complementary to that of the mRNA. Those skilledin the art also use the term “cDNA” to refer to a double-stranded DNAmolecule that comprises such a single-stranded DNA molecule and itscomplementary DNA strand.

Various methods are provided for detecting the presence of EGFR T790Mmutation contained in a sample (cancer tissue biopsy, cancer cellsobtained by laser tissue capture from a biopsy or cancer cells isolatedfrom serum). Such methods can include contacting a DNA sample with twoprimers that are upstream and downstream of the EGFR T790M region,amplifying the EGFR T790M region according to standard procedures, anddetecting whether the amplified sequence is present or absent in thenucleic acid sample. Accordingly, primers capable of recognizing andbinding to EGFR T790M upstream and downstream region and nucleic acidprobes having an affinity to EGFR T790M mutation are preferred means ofsupporting such methods. For example, the whole EGFR exon 20 can beamplified by PCR using genomic DNA as template and using primer pairscapable of recognizing and binding, respectively, to the 5′ and 3′intron flanking sequences of exon 20 (such exon 20 flanking sequencesare indicated with capital letters in SED ID NO:3 (Table 3, see GenBankAcc. No NT_(—)033968). Such primer pairs that include nucleotide 2369 ofthe EGFR cDNA sequence can amplify a fragment that it can then be usedfor sequencing, restriction length polymorphism analysis or any othertechnique for determining the presence or absence of the 2369 C→Tmutation.

TABLE 3 (SEQ ID NO: 3) 161101TTTAGCTTCC TCAGCCCAAG AATAGCAGAA GGGTTAAAAT AAAGTCTGTA TTTATGGCTC 161161TGTCAAAGGA AGGCCCCTGC CTTGGCAGCC AGCCGGAATT AGCAGGGCAG CAGATGCCTG 161221ACTCAGTGCA GCATGGATTT CCCATAGGGA GCCTGGGGGC ACAGCACAGA GAGACCACTT 161281CTCTTTAGAA ATGGGTCCCG GGCAGCCAGG CAGCCTTTAG TCACTGTAGA TTGAATGCTC 161341TGTCCATTTC AAAACCTGGG ACTGGTCTAT TGAAAGAGCT TATCCAGCTA CTCTTTGCAG 161401AGGTGCTGTG GGCAGGGTCC CCAGCCCAAA TGCCCACCCA TTTCCCAGAG CACAGTCAGG 161461GCCAAGCCTG GCCTGTGGGG AAGGGAGGCC TTTCTCCCTG CTGGCTCGGT GCTCCCCGGA 161521TGCCTTCTCC ATCGCTTGTC CTCTGCAGCA CCCACAGCCA GCGTTCCTGA TGTGCAGGGT 161581CAGTCATTAC CCAGGGTGTT CCGGACCCCA CACAGATTCC TACAGGCCCT CATGATATTT 161641TAAAACACAG CATCCTCAAC CTTGAGGCGG AGGTCTTCAT AACAAAGATA CTATCAGTTC 161701CCAAACTCAG AGATCAGGTG ACTCCGACTC CTCCTTTATC CAATGTGCTC CTCATGGCCA 161761CTGTTGCCTG GGCCTCTCTG TCATGGGGAA TCCCCAGATG CACCCAGGAG GGGCCCTCTC 161821CCACTGCATC TGTCACTTCA CAGCCCTGCG TAAACGTCCC TGTGCTAGGT CTTTTGCAGG 161881CACAGCTTTT CCTCCATGAG TACGTATTTT GAAACTCAAG ATCGCATTCA TGCGTCTTCA 161941CCTGGAAGGG GTCCATGTGC CCCTCCTTCT GGCCACCATG CGAAGCCACA CTGACGTGCC 162001TCTCCCTCCC TCCAGgaagc ctacgtgatg gccagcgtgg acaaccccca cgtgtgccgc 162061ctgctgggca tctgcctcac ctccaccgtg cagctcatca cgcagctcat gcccttcggc 162121tgcctcctgg actatgtccg ggaacacaaa gacaatattg gctcccagta cctgctcaac 162181tggtgtgtgc agatcgcaaa gGTAATCAGG GAAGGGAGAT ACGGGGAGGG GAGATAAGGA 162241GCCAGGATCC TCACATGCGG TCTGCGCTCC TGGGATAGCA AGAGTTTGCC ATGGGGATAT 162301GTGTGTGCGT GCATGCAGCA CACACACATT CCTTTATTTT GGATTCAATC AAGTTGATCT 162361TCTTGTGCAC AAATCAGTGC CTGTCCCATC TGCATGTGGA AACTCTCATC AATCAGCTAC 162421CTTTGAAGAA TTTTCTCTTT ATTGAGTGCT CAGTGTGGTC TGATGTCTCT GTTCTTATTT 162481CTCTGGAATT CTTTGTGAAT ACTGTGGTGA TTTGTAGTGG AGAAGGAATA TTGCTTCCCC 162541CATTCAGGAC TTGATAACAA GGTAAGCAAG CCAGGCCAAG GCCAGGAGGA CCCAGGTGAT 162601AGTGGTGGAG TGGAGCAGGT GCCTTGCAGG AGGCCCAGTG AGGAGGTGCA AGGAGCTGAC 162661AGAGGGCGCA GCTGCTGCTG CTATGTGGCT GGGGCCTTGG CTAAGTGTCC CCCTTTCCAC 162721AGGCTCGCTC CAGAGCCAGG GCGGGGCTGA GAGAGCAGAG TGGTCAGGTA GCCCTGCCTG 162781GGTGCTGGAG ACAGGCACAG AACAACAAGC CAGGTATTTC ACAGCTGGTG CGGACCCAGA 162841AAGACTTCTG CTTTTGCCCC AAACCCCTCC CATCTCCATC CCAGTCTTGC ATCAGTTATT 162901TGCACTCAAC TTGCTAAGTC CTATTTTTTT CTAACAATGG GTATACATTT CATCCCATTG 162961ACTTTAAAGG ATTTGCAGGC AGGCCCTGTC TCTGAGAATA CGCCGTTGCC CGTCATCTCT 163021CTCCGACAGC AGGGCAGGGG GTCCAGAGAT GTGCCAGGGA CCAGAGGGAG GGAGCAGACA 163081CCCACCCGGC CTGGGCAGGT CCTCCTCATT GCTTGCATCC GCCTGGTTAG CAGTGGCAGT 163141CAGTCCTGCC GAGTCATTCG TGAGGCGCTC ACCCAACTCC AGGCAGATGT AAAAGGTGAC 163201CTACAAGAAG ACAAACAAAA ACATCTGGAG CGCTCTTATG CCAGCATCTG CCCTTGACAC

Without limiting to these diagnostic methods, a method is provided fordetecting EGFR T790M mutation whereby a restriction enzyme is used torecognize the lack or presence of a restriction site at the alleliccodon. A restriction site leading to productive cleaving of thepolynucleotide occurs, using a suitably selective restriction nuclease,when one or the other of the wild type or the polymorphic allele ispresent.

Also envisioned in the present invention is a diagnostic kit fordetecting mutant EGFR T790M related malignancy in a human. Such a kitpreferably includes multiple containers wherein included is a set ofprimers useful for PCR detection of the EGFR T790M mutation, andoptionally a positive control comprising mutated EGFR sequence and anegative control comprising a non-mutated EGFR sequence.

FIG. 2 provides schematic representations of certain embodiments of theprimers of the invention. The invention discloses sequences that serveas primers to amplify segments of genomic or cDNA sequences of EGFR thatinclude the base corresponding to position 2369 of EGFR cDNA. Thedisclosed primer sequences, such as SEQ ID NOS:4-7 and 12-15, arerepresented schematically by the lightly shaded blocks in FIG. 2. FIG.2, a) illustrates an embodiment in which the disclosed primer shown as“SEQ” may optionally be included in a larger polynucleotide whoseoverall length may range up to 200 nucleotides.

The invention further provides a primer sequence that is a fragment ofany of the above primer sequences, SEQ ID NOS:4-7 and 12-15, that is atleast 11 nucleotides in length (and at most 1 base shorter than thereference SEQ ID NO: illustrated in FIG. 2, b)), as well as a primersequence wherein up to 5 nucleotides may differ from the sequences givenin SEQ ID NOS:4-7 and 12-15 (illustrated in FIG. 2, c), showing, in thisexample, three variant bases represented by the three darker verticalbars).

Still further the invention provides a sequence that is a complement toany of the above-described sequences (shown in FIG. 2, d), anddesignated as “COMPL”). Any of these sequences are included in theoligonucleotides or polynucleotides of the invention. As noted, anyprimer polynucleotide of the invention optionally may include additionalbases up to the limit of 200 nucleotides.

Primers of the invention are designed to be “substantially”complementary to each strand of the genomic locus or cDNA to beamplified. This means that the primers must be sufficientlycomplementary to hybridize with their respective strands underconditions which allow the polymerase chain reaction to proceed. Inother words, the primers should have sufficient complementarity with the5′ and 3′ sequences flanking the mutation to hybridize therewith andpermit amplification of the genomic locus. Thus it is envisioned hereinthat a primer sequence need not be fully complementary to its targetsequence. “Substantially identical” and similar phrases that refer tooligonucleotide sequences thus describes the functional ability tohybridize or anneal with sufficient specificity to distinguish betweenthe presence or absence of a mutation, such as a SNP identified herein.This is measurable by the temperature of melting being sufficientlydifferent to permit easy identification of whether the oligonucleotideis binding to the normal or mutant EGFR T790M gene sequence.Oligonucleotide primers of the invention are employed in theamplification process which is an enzymatic chain reaction that producesexponential quantities of polymorphic locus relative to the number ofreaction steps involved. Typically, one primer is complementary to thenegative (−) strand of the polymorphic locus and the other iscomplementary to the positive (+) strand. Annealing the primers todenatured nucleic acid followed by extension with an enzyme, DNApolymerase, and nucleotides, results in newly synthesized + and −strands containing the target polymorphic locus sequence. Because thesenewly synthesized sequences are also templates, repeated cycles ofdenaturing, primer annealing, and extension results in exponentialproduction of the region (i.e., the target polymorphic locus sequence)defined by the primers. The product of the chain reaction is a discreetnucleic acid duplex with termini corresponding to the ends of thespecific primers employed.

Synthesis of Polynucleotides.

The oligonucleotide primers of the invention may be prepared using anysuitable method, such as conventional phosphotriester and phosphodiestermethods or automated embodiments thereof. In one such automatedembodiment, diethylphosphoramidites are used as starting materials andmay be synthesized as described by Beaucage, et al., TetrahedronLetters, 22:1859-1862, (1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066.

Oligonucleotides and polynucleotides can be prepared by standardsynthetic techniques, e.g., using an automated DNA synthesizer. Methodsfor synthesizing oligonucleotides include well-known chemical processes,including, but not limited to, sequential addition of nucleotidephosphoramidites onto surface-derivatized particles, as described by T.Brown and Dorcas J. S. Brown in Oligonucleotides and Analogues APractical Approach, F. Eckstein, editor, Oxford University Press,Oxford, pp. 1-24 (1991), and incorporated herein by reference.

An example of a synthetic procedure uses Expedite RNA phosphoramiditesand thymidine phosphoramidite (Proligo, Germany). Syntheticoligonucleotides are deprotected and gel-purified (Elbashir et al.(2001) Genes & Dev. 15, 188-200), followed by Sep-Pak C18 cartridge(Waters, Milford, Mass., USA) purification (Tuschl et al. (1993)Biochemistry, 32:11658-11668). Other methods of oligonucleotidesynthesis include, but are not limited to solid-phase oligonucleotidesynthesis according to the phosphotriester and phosphodiester methods(Narang, et al., (1979) Meth. Enzymol. 68:90), and to the H-phosphonatemethod (Garegg, P. J., et al., (1985) “Formation of internucleotidicbonds via phosphonate intermediates”, Chem. Scripta 25, 280-282; andFroehler, B. C., et al., (1986a) “Synthesis of DNA via deoxynucleosideH-phosphonate intermediates”, Nucleic Acid Res., 14, 5399-5407, amongothers) and synthesis on a support (Beaucage, et al. (1981) TetrahedronLetters 22:1859-1862) as well as phosphoramidate techniques (Caruthers,M. H., et al., “Methods in Enzymology,” Vol. 154, pp. 287-314 (1988),U.S. Pat. Nos. 5,153,319, 5,132,418, 4,500,707, 4,458,066, 4,973,679,4,668,777, and 4,415,732, and others described in “Synthesis andApplications of DNA and RNA,” S. A. Narang, editor, Academic Press, NewYork, 1987, and the references contained therein, and nonphosphoramiditetechniques. Solid phase synthesis helps isolate the oligonucleotide fromimpurities and excess reagents. Once cleaved from the solid support theoligonucleotide may be further isolated by known techniques.

Any nucleic acid specimen, in purified or nonpurified form, can beutilized as the starting nucleic acid(s), providing it contains, or issuspected of containing, the specific nucleic acid sequence containingthe polymorphic locus. Thus, the process may amplify, for example, DNAor RNA, including messenger RNA, wherein DNA or RNA may be singlestranded or double stranded. In the event that RNA is to be used as atemplate, enzymes, and/or conditions optimal for reverse transcribingthe template to cDNA would be utilized. In addition, a DNA-RNA hybridwhich contains one strand of each may be utilized. A mixture of nucleicacids may also be employed, or the nucleic acids produced in a previousamplification reaction herein, using the same or different primers maybe so utilized. The specific nucleic acid sequence to be amplified,i.e., the polymorphic locus, may be a fraction of a larger molecule orcan be present initially as a discrete molecule, so that the specificsequence constitutes the entire nucleic acid. It is not necessary thatthe sequence to be amplified be present initially in a pure form; it maybe a minor fraction of a complex mixture, such as contained in wholehuman DNA.

DNA utilized herein may be extracted from a body sample, tissue materialand the like by a variety of techniques such as that described byManiatis, et. al. in Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., p 280-281, 1982). If the extracted sample is impure, itmay be treated before amplification with an amount of a reagenteffective to open the cells, or animal cell membranes of the sample, andto expose and/or separate the strand(s) of the nucleic acid(s). Thislysing and nucleic acid denaturing step to expose and separate thestrands will allow amplification to occur much more readily.

The agent for DNA polymerization may be any compound or system whichwill function to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose include, forexample, polymerase muteins, reverse transcriptase, other enzymes,including heat-stable enzymes (i.e., those enzymes which perform primerextension after being subjected to temperatures sufficiently elevated tocause denaturation), such as Taq polymerase. The suitable enzyme willfacilitate combination of the nucleotides in the proper manner to formthe primer extension products which are complementary to eachpolymorphic locus nucleic acid strand. Generally, the synthesis will beinitiated at the 3′ end of each primer and proceed in the 5′ directionalong the template strand, until synthesis terminates, producingmolecules of different lengths.

The amplification products may be detected by analyzing it by Southernblots without using radioactive probes. In such a process, for example,a small sample of DNA containing a very low level of the nucleic acidsequence of the polymorphic locus is amplified, and analyzed via aSouthern blotting technique or similarly, using dot blot analysis. Theuse of non-radioactive probes or labels is facilitated by the high levelof the amplified signal. Alternatively, probes used to detect theamplified products can be directly or indirectly detectably labeled, forexample, with a radioisotope, a fluorescent compound, a bioluminescentcompound, a chemiluminescent compound, a metal chelator or an enzyme.Those of ordinary skill in the art will know of other suitable labelsfor binding to the probe, or will be able to ascertain such, usingroutine experimentation. In the preferred embodiment, the amplificationproducts are determinable by separating the mixture on an agarose gelcontaining ethidium bromide which causes DNA to be fluorescent.

Sequences amplified by the methods of the invention can be furtherevaluated, detected, cloned, sequenced, and the like, either in solutionor after binding to a solid support, by any method usually applied tothe detection of a specific DNA sequence such as PCR, oligomerrestriction (Saiki, et. al., Bio/Technology, 3:1008-1012, (1985)),allele-specific oligonucleotide (ASO) probe analysis (Conner, et. al.,Proc. Natl. Acad. Sci. U.S.A., 80:278, (1983)), oligonucleotide ligationassays (OLAs) (Landgren, et. al., Science, 241:1007, (1988)), and thelike. Molecular techniques for DNA analysis have been reviewed(Landgren, et. al., Science, 242:229-237, (1988)).

Preferably, the method of amplifying is by PCR, as described herein andas is commonly used by those of ordinary skill in the art. Alternativemethods of amplification have been described and can also be employed aslong as the EGFR locus amplified by PCR using primers of the inventionis similarly amplified by the alternative means. Such alternativeamplification systems include but are not limited to self-sustainedsequence replication, which begins with a short sequence of RNA ofinterest and a T7 promoter. The enzyme reverse transcriptase copies RNAinto cDNA followed by degradation of the transcribed RNA. Anothernucleic acid amplification technique is nucleic acid sequence-basedamplification (NASBA) which uses reverse transcription and T7 RNApolymerase and incorporates two primers to target its cycling scheme.NASBA can begin with either DNA or RNA and finish with either, andamplifies to 10⁸ copies within 60 to 90 minutes. Alternatively, nucleicacid can be amplified by ligation activated transcription (LAT). LATworks from a single-stranded template with a single primer that ispartially single-stranded and partially double-stranded Amplification isinitiated by ligating a cDNA to the promoter oligonucleotide and withina few hours, amplification is 10⁸ to 10⁹ fold. The QB replicase systemcan be utilized by attaching an RNA sequence called MDV-1 to RNAcomplementary to a DNA sequence of interest. Upon mixing with a sample,the hybrid RNA finds its complement among the specimen's mRNAs andbinds, activating the replicase to copy the tag-along sequence ofinterest. Another nucleic acid amplification technique, ligase chainreaction (LCR), works by using two differently labeled halves of asequence of interest which are covalently bonded by ligase in thepresence of the contiguous sequence in a sample, forming a new target.The repair chain reaction (RCR) nucleic acid amplification techniqueuses two complementary and target-specific oligonucleotide probe pairs,thermostable polymerase and ligase, and DNA nucleotides to geometricallyamplify targeted sequences. A 2-base gap separates the oligonucleotideprobe pairs, and the RCR fills and joins the gap, mimicking normal DNArepair. Nucleic acid amplification by strand displacement activation(SDA) utilizes a short primer containing a recognition site for Hinc IIwith short overhang on the 5′ end which binds to target DNA. A DNApolymerase fills in the part of the primer opposite the overhang withsulfur-containing adenine analogs. Hinc II is added but only cuts theunmodified DNA strand. A DNA polymerase that lacks 5′ exonucleaseactivity enters at the cite of the nick and begins to polymerize,displacing the initial primer strand downstream and building a new onewhich serves as more primer. SDA produces greater than 10.sup.7-foldamplification in 2 hours at 37 degree C. Unlike PCR and LCR, SDA doesnot require instrumented Temperature cycling. Another amplificationsystem useful in the method of the invention is the QB Replicase System.Although PCR is the preferred method of amplification in the invention,these other methods can also be used to amplify the locus as describedin the method of the invention.

A variety of methods well-known in the art can be used for detection ofpredetermined sequence variations by allele specific hybridization.Preferably, the test gene is probed with allele specificoligonucleotides (ASOs); and each ASO contains the sequence of a knownmutation. ASO analysis detects specific sequence variations in a targetpolynucleotide fragment by testing the ability of a specificoligonucleotide probe to hybridize to the target polynucleotidefragment. Preferably, the oligonucleotide contains the mutant sequence(or its complement). The presence of a sequence variation in the targetsequence is indicated by hybridization between the oligonucleotide probeand the target fragment under conditions in which an oligonucleotideprobe containing a normal sequence does not hybridize to the targetfragment. A lack of hybridization between the sequence variant (e.g.,mutant) oligonucleotide probe and the target polynucleotide fragmentindicates the absence of the specific sequence variation (e.g.,mutation) in the target fragment. In a preferred embodiment, the testsamples are probed in a standard dot blot format. Each region within thetest gene that contains the sequence corresponding to the ASO isindividually applied to a solid surface, for example, as an individualdot on a membrane. Each individual region can be produced, for example,as a separate PCR amplification product using methods well-known in theart (see, for example, the experimental embodiment set forth in Mullis,K. B., 1987, U.S. Pat. No. 4,683,202).

Membrane-based formats that can be used as alternatives to the dot blotformat for performing ASO analysis include, but are not limited to,reverse dot blot, (multiplex amplification assay), and multiplexallele-specific diagnostic assay (MASDA).

In a reverse dot blot format, oligonucleotide or polynucleotide probeshaving known sequence are immobilized on the solid surface, and aresubsequently hybridized with the labeled test polynucleotide sample. Inthis situation, the primers may be labeled or the NTPs maybe labeledprior to amplification to prepare a labeled test polynucleotide sample.Alternatively, the test polynucleotide sample may be labeled subsequentto isolation and/or synthesis In a multiplex format, individual samplescontain multiple target sequences within the test gene, instead of justa single target sequence. For example, multiple PCR products eachcontaining at least one of the ASO target sequences are applied withinthe same sample dot. Multiple PCR products can be producedsimultaneously in a single amplification reaction using the methods ofCaskey et al., U.S. Pat. No. 5,582,989. The same blot, therefore, can beprobed by each ASO whose corresponding sequence is represented in thesample dots.

A MASDA format expands the level of complexity of the multiplex formatby using multiple ASOs to probe each blot (containing dots with multipletarget sequences). This procedure is described in detail in U.S. Pat.No. 5,589,330 by A. P. Shuber, and in Michalowsky et al., AmericanJournal of Human Genetics, 59(4): A272, poster 1573 (October 1996), eachof which is incorporated herein by reference in its entirety. First,hybridization between the multiple ASO probe and immobilized sample isdetected. This method relies on the prediction that the presence of amutation among the multiple target sequences in a given dot issufficiently rare that any positive hybridization signal results from asingle ASO within the probe mixture hybridizing with the correspondingmutant target. The hybridizing ASO is then identified by isolating itfrom the site of hybridization and determining its nucleotide sequence.

Designing an Allele Specific Oligonucleotide (ASO) Probe

An allele specific oligonucleotide probe is a short, single strandedpolynucleotide that is engineered to hybridize exactly to a targetsequence under a given set of conditions. Routinely, ASO probes aredesigned to contain sequences identical to the normal allele andsequence variation respectively. Hybridization of the probe to thetarget allows for the discrimination of a variant sample. Understringent conditions, a probe with a variation as simple as asingle-base pair will not hybridize to a normal sequence due to adestabilizing effect of the normal-mutant duplex (Ikuta, S. et al,Nucleic Acids Research, 15: 797-811 (1987).

The design of an ASO hybridization probe must meet two basicrequirements. (Current Protocols in Human Genetics, section 9.4,(1995)). First, probes that are used together in the same pool should bearound the same length. Although the standard length of a probe isoptimally 17 base pairs, the range can be as short as about 14 or aslong as about 27. If the mutation contains a long insertion, a longerprobe may be desirable. Second, the mismatched region should not beplaced at the end of the 17 base pair probe, but approximately in themiddle of the sequence, approximately 5-7 bases from the 5′ end of theprobe. In addition, the placement of a mismatch, in the case of a longerprobe, should not be at the end, but at a position that allows stronghybridization and stabilization of the polynucleotide strand. In orderto minimize the effects of variations in base composition of the probes,tetramethylammonium chloride is used as in the ASO hybrid's buffer(Shuber, T., U.S. Pat. No. 5,633,134). Conventionally, ASO probes aresynthesized on a DNA synthesizer. They can be labeled with isotopic ornon-isotopic detection agents using means familiar to those of skill inthe art. The process outlined in this application for making and usingprobes can be applicable for other gene sequences.

Suitable materials that can be used in the dot blot, reverse dot blot,multiplex, and MASDA formats are well-known in the art and include, butare not limited to nylon and nitrocellulose membranes.

When the target sequences are produced by PCR amplification, thestarting material can be chromosomal DNA in which case the DNA isdirectly amplified. Alternatively, the starting material can be mRNA, inwhich case the mRNA is first reversed transcribed into cDNA and thenamplified according to the well known technique of RT-PCR (see, forexample, U.S. Pat. No. 5,561,058 by Gelfand et al.).

The methods described above are suitable for moderate screening of alimited number of sequence variations. However, with the need inmolecular diagnosis for rapid, cost effective large scale screening,technologies have developed that integrate the basic concept of ASO, butfar exceed the capacity for mutation detection and sample number. Thesealternative methods to the ones described above include, but are notlimited to, large scale chip array sequence-based techniques. The use oflarge scale arrays allows for the rapid analysis of many sequencevariants. A review of the differences in the application and developmentof chip arrays is covered by Southern, E. M., Trends In Genetics, 12:110-115 (March 1996) and Cheng et al., Molecular Diagnosis, 1:183-200(September 1996). Several approaches exist involving the manufacture ofchip arrays. Differences include, but not restricted to: type of solidsupport to attach the immobilized oligonucleotides, labeling techniquesfor identification of variants and changes in the sequence-basedtechniques of the target polynucleotide to the probe.

A promising methodology for large scale analysis on ‘DNA chips’ isdescribed in detail in Hacia et al., Nature Genetics, 14:441-447 (1996),which is hereby incorporated by reference in its entirety. As describedin Hacia et al., high density arrays of over 96,000 oligonucleotides,each 20 nucleotides in length, are immobilized to a single glass orsilicon chip using light directed chemical synthesis. Contingent on thenumber and design of the oligonucleotide probe, potentially every basein a sequence can be interrogated for alterations. Oligonucleotidesapplied to the chip, therefore, can contain sequence variations that arenot yet known to occur in the population, or they can be limited tomutations that are known to occur in the population.

Prior to hybridization with oligonucleotide probes on the chip, the testsample is isolated, amplified and labeled (e.g. fluorescent markers) bymeans well known to those skilled in the art. The test polynucleotidesample is then hybridized to the immobilized oligonucleotides. Theintensity of sequence-based techniques of the target polynucleotide tothe immobilized probe is quantitated and compared to a referencesequence. The resulting genetic information can be used in moleculardiagnosis.

In another embodiment of the invention, a method is provided fordiagnosing the underlying cause for a subject having a relapse incancer, or a relapse in lung cancer, comprising sequencing a targetnucleic acid of a sample from a subject following amplification of thetarget nucleic acid. The EGFR gene, or fragments thereof, may be clonedand then sequenced to determine the presence of absence of a mutation.In such a situation, one need only compare the sequence obtained to anaturally occurring (wild type) EGFR gene, or portion thereof.

Other methods of DNA sequencing such as those of Sanger et al, Proc.Natl. Acad. Sci. USA, 74: 5463 (1977) or Maxam et al, Proc. Natl. Acad.Sci. USA, 74: 560 (1977) or other methods known in the art may be used.

In another embodiment of the invention a method is provided fordiagnosing the underlying cause for a subject having a relapse in cancercomprising contacting a target nucleic acid of a sample from a subjectwith a reagent that detects the presence of the mutation of the presentinvention and detecting the mutation.

Another method comprises contacting a target nucleic acid of a samplefrom a subject with a reagent that detects the presence of the mutationand detecting the mutation. A number of hybridization methods are wellknown to those skilled in the art. Many of them are useful in carryingout the invention.

The materials for use in the method of the invention are ideally suitedfor the preparation of a diagnostic kit. Such a kit may comprise acarrier means being compartmentalized to receive in close confinementone or more container means such as vials, tubes, and the like, each ofthe container means comprising one or more of the separate elements tobe used in the method. For example, one of the container means maycomprise means for amplifying EGFR DNA, or a fragment thereof, saidmeans comprising the necessary enzyme(s) and oligonucleotide primers foramplifying said target DNA from the subject. Another container maycontain oligonucleotide probes for detecting the presence or absence ofa mutation. Alternatively, another container may contain a restrictionenzyme that recognizes the mutant sequence but not the wild type, orvice versa.

Other methods can include contacting a cancer tissue sample from acancer patient with an antibody that specifically detects the EGFR T790Mform of the EGFR protein but not the EFGR protein not containing thismutation. Alternatively a protein extract from a cancer tissue samplefrom a cancer patient can be obtained and analyzed by western blot,ELISA, or other protein detection techniques, for the presence orabsence of the EGFR T790M mutant using an antibody specific to detectthis mutation and not the EGFR protein not containing this mutation. Theantibody to detect EGFR T790M mutant can be an antibody obtained from ahybridoma. A typical procedure for making hybridomas is as follows: (a)immunize mice with a certain immunogen; (b) remove the spleens from theimmunized mice and make a spleen suspension in an appropriate medium;(c) fuse the suspended spleen cells with mouse myeloma cells; (d) diluteand culture the mixture of unfused spleen cells, unfused myeloma cellsand fused cells in a selective medium which will not support growth ofthe unfused myeloma cells or spleen cells; (e) evaluate the supernatantin each container containing hybridoma for the presence of antibody tothe immunogen; and (f) select and clone hybridomas producing the desiredantibodies. Once the desired hybridoma has been selected and cloned, theresultant antibody is produced by in vitro culturing of the desiredhybridoma in a suitable medium. As an alternative method, the desiredhybridoma can be injected directly into mice to yield concentratedamounts of antibody [Kennett, et al., (1981) Ed., Monoclonal Antibodies.Hybridomas: A new dimension in biological analyses, Plenum Press, NewYork]. Hybridomas produced by fusion of murine spleen cells and murinemyeloma cells have been described in the literature by Kohler et al., inEur. J. Immunol. 6, 511-519 (1976); by Milstein et al. in Nature, 266,550 (1977); and by Walsh, Nature, 266, 550 (1977); and by Walsh, Nature,266, 495 (1977). The technique is also set out in some detail byHerzenberg and Milstein, in Handbook on Experimental Immunology, ed.Weir (Blackwell Scientific, London), 1979, pages 25.1 to 25.7 as well asin Kennett et al., supra. Patents relating to monoclonal antibodiesagainst human tumors produced by hybridoma technology include U.S. Pat.Nos. 4,182,124 and 4,196,265. Representative of the art concerningmonoclonal antibodies that have specificity for antigens on carcinomacells are U.S. Pat. No. 4,350,683.

Specific mutations in the tyrosine kinase domain of EGFR are associatedwith sensitivity to either gefitinib or erlotinib, but mechanisms ofacquired resistance have not yet been reported. Based upon analogousstudies in other diseases with another kinase inhibitor, imatinib, asingle amino acid substitution from threonine to methionine at position790 in the wild-type EGFR kinase domain was predicted to lead to drugresistance, even before the association of exon 19 and 21 mutations ofEGFR with drug responsiveness in NSCLC was reported. The C to Ttransition replacing Thr-766 with methionine (ACG to ATG) mutation wasshown in vitro in the context of wild-type EGFR to confer resistance togefitinib [21] and a related quinazoline inhibitor, PD153035 [22].

EXAMPLES Materials and Methods

Tissue Procurement

Tumor specimens, including paraffin blocks, fine needle biopsies, andpleural effusions, were obtained through protocols approved by theInstitutional Review Board of Memorial Sloan-Kettering Cancer Center(protocol 92-055 [7] and protocol 04-103 [Protocol S1]). All patientsprovided informed consent.

Mutational Analyses of EGFR and KRAS in Lung Tumors

Genomic DNA was extracted from tumor specimens, and primers for EGFR(exons 18-24) and KRAS2 (exon 2) analyses were as published [3,7]. Allsequencing reactions were performed in both forward and reversedirections, and all mutations were confirmed at least twice fromindependent PCR isolates.

The exon 20 mutation (T790M) was also detected by length analysis offluorescently labeled (FAM) PCR products on a capillary electrophoresisdevice (ABI 3100 Avant, Applied Biosystems, Foster City, Calif., UnitedStates), based on a new NlaIII restriction site created by the T790Mmutation (2369 C→T). The following primers were used:

EGFR Ex20F, 5′-FAM-CTCCCTCCAGGAAGCCTACGTGAT-3′ (SEQ ID NO:4) and

EGFR Ex20R 5′-TTTGCGATCTGCACACACCA-3′ (SEQ ID NO:5). Using seriallymixed dilutions of DNA from NSCLC cell lines (H1975, L858R- andT790M-positive; H-2030, EGFR wild-type) for calibration, this assaydetects the presence of the T790M mutation when H1975 DNA comprises 3%or more of the total DNA tested, compared to a sensitivity of 6% fordirect sequencing (data not shown), with the caveat that the allelecontaining the T790M mutation is amplified about 2-fold in H1975 cells.

RT-PCR

The following primers were used to generate EGFR cDNA fragments spanningexon 20:

EGFR 2095F 5′-CCCAACCAAGCTCTCTTGAG-3′ (SEQ ID NO:6) and

EGFR 2943R 5′-ATGACAAGGTAGCGCTGGGGG-3′ (SEQ ID NO:7). The sequencestargeted by EGFR 2095F and EGFR 2943R are shown underlined in Table 1.PCR products were ligated into plasmids using the TOPO TA-cloning kit(Invitrogen, Carlsbad, Calif., United States), as per manufacturer'sinstructions Minipreps of DNA from individual clones were sequencedusing the T7 priming site of the cloning vector.

Functional Analyses of Mutant EGFRs

Mutations were introduced into full-length wild-type and mutant EGFRcDNAs using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif., United States) and cloned into expression vectors asdescribed [3]. The following primers were used to generate the deletion(del) L747−E749;A750P mutant:

-   -   forward 5′-TAAAATTCCCGTCGCTATCAAGGAGCCAACATCTCCGAAA        GCCAACAAGG-3′ (SEQ ID NO:8) and    -   reverse 5′-CCTTGTTGGCTTTCGGAGATGTTGGCTCCTTGATAGCGACG        GGAATTTTA-3′ (SEQ ID NO:9). The following primers were used to        introduce the T790M mutation:    -   forward 5′-AGCTCATCATGCAGCTCAT-3′ (SEQ ID NO:10) and    -   reverse 5′-ATGAGCTGCATGATGAGCT-3′ (SEQ ID NO:11). The L858R        mutant cDNA was generated previously [3]. All mutant clones were        fully re-sequenced bidirectionally to ensure that no additional        mutations were introduced. Various EGFRs were transiently        expressed in 293T human embryonic kidney cells as published [3].        Cells were treated with different concentrations of gefitinib or        erlotinib.        Immunoblotting

See methods and supplementary methods in [3] for details on cell lysis,immunoblotting, and antibody reagents. At least three independentexperiments were performed for all analyses.

Cell Culture

The NSCLC cell lines H1650, H1975, H2030, H2347, H2444, H358, and H1734were purchased from American Type Culture Collection (Manassas, Va.,United States). H3255 was a gift of B. Johnson and P. Janne. Cells weregrown in complete growth medium (RPMI-1640; American Type CultureCollection catalog no. 30-2001) supplemented with 10% fetal calf serum,10 units/ml penicillin, and 10 μg/ml streptomycin) at 37° C. and 5% CO₂.For viability studies, cells were seeded in complete growth medium inblack 96-well clear bottom VIEWPLATES (PerkinElmer, Wellesley, Mass.,United States) at a density of 5,000 (H1975 and H2030) or 7,500 cellsper well (H3255). Following overnight incubation, cells were grown for24 h in the supplemented RPMI-1640 medium with 0.1% serum. Cells (insupplemented RPMI-1640 medium containing 0.1% serum) were then incubatedfor 48 h in the continued presence of gefitinib or erlotinib.

Viability Assay

Cell viability was assayed using Calcein AM (acetoxymethyl ester ofCalcein, Molecular Probes, Eugene, Oreg., United States). Followingincubation with gefitinib or erlotinib, monolayers were washed twicewith PBS (containing calcium and magnesium) and incubated with 7.5 μmolCalcein AM in supplemented RPMI-1640 (no serum) for 30 min. Labelingmedium was removed, and cells were washed three times with PBS. Calceinfluorescence (Ex, 485 nm; Em, 535 nM) was detected immediately using aVICTOR™ V multi-label plate reader (PerkinElmer). Three independentexperiments were performed for each cell line; each experiment includedfour to eight replicates per condition.

Production of Anti-Mutant EGFR Monoclonal and Polyclonal Antibodies

A group of three Balb/c female mice (Charles River BreedingLaboratories, Wilmington, Mass.) are injected with 5 ug/dose of purifiedtruncated EGFR protein or fragment thereof containing the T790M mutationin 100 ul Detox adjuvant (RIBI ImmunoChem Res Inc, Hamilton, Mo.) byintraperitoneal injection on days 0, 3, 7, 10, and 14. On day 17 theanimals are sacrificed, their spleens are removed and the lymphocytesfused with the mouse myeloma line 653 using 50% polyethylene glycol 4000by an established procedure (see U.S. Pat. Nos. 5,939,269, and 5,658,791as incorporated herein by reference). The fused cells are plated into96-well microtiter plates at a density of 2×10⁵ cells/well followed byHAT selection on day 1 post-fusion. Immobilized hybridoma culturesupernatants are then reacted with biotinylated EGFR T790M mutant. Thewells positive for anti-EGFR antibodies are expanded for further study.These cultures remain stable when expanded and cell lines arecryopreserved. The parental cultures are isotyped and then assayed fortheir ability to capture and to specifically recognize EGFR T790Mmutant.

Alternatively, polyclonal rabbit antisera is raised against purifiedmutant protein peptides Polyclonal antibodies against the EGFR T790Mmutant are obtained by coupling such peptides to Keyhole LimpetHemocyanin with 0.05% glutaraldehyde, emulsified in Freund's completeadjuvant and injected intradermally at several sites. The animals areboosted four and seven weeks later with coupled peptide emulsified inFreund's incomplete adjuvant and bled ten days after the last injection.

Antibodies prepared according to the above procedures are then used foridentifying and/or diagnosing tumor cells (i.e. in ultrathin sections ofcancer tissues) for expression of EGFR T790M mutation and/or fortherapeutic approaches according to standard procedures known in theart, e.g., U.S. Pat. Nos. 5,601,989, 5,563,247, 5,610,276, and5,405,941, as incorporated herein by way of reference. These sameantibodies are used for monitoring expression of EGFR T790M mutant.

Example 1

To confirm the presence of the EGFR T790M mutation, an allele-specificoligonucleotide PCR based assay (Guo, Z., Liu, Q. & Smith, L. M.Enhanced discrimination of single nucleotide polymorphisms by artificialmismatch hybridization. Nat. Biotechnol. 15, 331-335 (1997)) isperformed by amplifying the mutant allele using one base mismatch PCRprimers containing one 3′ end and a 3-nitropyrrole residue. PCR productsare created with a 3′ mutant allele specific primer

(5′ CACCGTGCAGCTCATCAT 3′ (SEQ ID NO: 12) or 5′ CGAAGGGCATGAGCTGCG 3′(SEQ ID NO: 13))containing the complement to the mutant base at the 3′ end and a3-nitropyrrole residue upstream of the 3′ end. The mutant allelespecific primer is capable of amplifying mutant DNA derived from frozenor paraffin-embedded tumors, but is unable to produce a product fromnormal DNA. At the same time, a wild-type (WT) 3′ primer

(5′ CACCGTGCAGCTCATCAC 3′ (SEQ ID NO: 14) or 5′ CGAAGGGCATGAGCTGCA 3′(SEQ ID NO: 15))is able to amplify only normal wild-type DNA but not mutant DNA. Theseexperiments show that the mutant allele is amplified in tumor samples,whereas it is not amplified in normal adjacent tissues.

Example 2 Clonal Origin of the EGFR T790M Mutation

When careful tumor microdissection is performed in attempt to increasethe relative percentage of tumor cells in any given sample, the ratio ofthe T:C alleles increases proportionately. The PCR is performed with oneof the primers described in Example 1 and another primer amplifying inthe contrary sense, so that a readily detectable fragment can beobtained for the EGFR sequence, either mutated or wild type, whosepresence is being sought. The sequence of such primer can be easilydesigned by those of ordinary skill in the art. Results of suchprocedures demonstrate that the EGFR T790M mutation is clonal in origin.

Example 3 Assay for the EGFR T790M Mutation in Genomic DNA

A method is provided for detecting EGFR T790M mutation whereby arestriction enzyme MaHI is used to recognize the lack or presence ofrestriction site at the mutated codon. In this Example an assay isprovided using a primer that spans the intron-exon boundary for exon 20.The fluorescence-based detection takes advantage of a PCR restrictionfragment length polymorphism (PCR-RFLP) generated by the specificmissense mutation. PCR amplification is performed with theexon-20-specific primers EGFR Ex20F (SEQ ID NO:4) and EGFR Ex20R (SEQ IDNO:5) (underlined in Table 4) spanning nucleotide 2369. Table 4 includesa portion (Seq ID No. 28) of the larger intron-exon 20-intron genomicsequence given in Table 3 (SEQ ID NO:3) from position 161904 to position162970. The 3′ terminus of the intron upstream from exon 20 is shown inbold type.

TABLE 4. . . gtattttgaaactcaagatcgcattcatgcgtcttcacctggaaggggtccatgtgcccctccttctggccaccatgcgaagccacactgacgtgcctctcc ctccctccaggaagcctacgtgatggccagcgtggacaacccccacgtgtgccgcctgctgggcatctgcctcacctccaccgtgcagctcatcacgcagctcatgcccttcggctgcctcctggactatgtccgggaacacaaagacaatattggctcccagtacctgctcaactggtgtgtgcagatcgcaaagg . . .

The wild-type sequence contains specific N1aIII sites, which upondigestion yield a 106-bp product (see Methods; FIG. 3A). Presence of themutant 2369 T nucleotide creates a new NlaIII restriction digest site,yielding a slightly shorter product (97 bp; FIG. 3A), which is readilydetected by fluorescent capillary electrophoresis. This test is about2-fold more sensitive than direct sequencing. Any equivalent means thatcleaves one of the 2369 alleles (wild type or mutant) but not the otheris contemplated to be useful in this labeled fragment-length assay. Theassay requires use of any known method of incorporating a label into thePCR amplicon such that resulting fragments are detectable.

Example 4 Case Reports

We identified secondary EGFR mutations in three of six individuals whosedisease progressed on either gefitinib or erlotinib (Table 5). Briefcase histories of these three patients are presented below.

Patient 1.

This 63-y-old female “never smoker” (smoked less than 100 cigarettes inher lifetime) initially presented with bilateral diffuse chest opacitiesand a right-sided pleural effusion. Transbronchial biopsy revealedadenocarcinoma. Disease progressed on two cycles of systemicchemotherapy, after which gefitinib, 250 mg daily, was started.Comparison of chest radiographs obtained prior to starting gefitinib(FIG. 4A, left panel) and 2 wk later (FIG. 4A, middle panel) showeddramatic improvement. Nine mo later, a chest radiograph revealedprogression of disease (FIG. 4A, right panel). Subsequently, the patientunderwent a computed tomography (CT)-guided biopsy of an area in theright lung base (FIG. 5A, left panel). Despite continued treatment withgefitinib, either with chemotherapy or at 500 mg daily, the pleuraleffusion recurred, 12 mo after initiating gefitinib (FIG. 5A, rightpanel). Pleural fluid was obtained for molecular studies. In total, thispatient had three tumor specimens available for analysis: the originallung tumor biopsy, a biopsy of the progressing lung lesion, and pleuralfluid. However, re-review of the original transbronchial biopsy showedthat it had scant tumor cells (Table 5).

Patient 2.

This 55-y-old woman with a nine pack-year history of smoking underwenttwo surgical resections within 2 y (right lower and left upperlobectomies) for bronchioloalveolar carcinoma with focal invasion. Twoyears later, her disease recurred with bilateral pulmonary nodules andfurther progressed on systemic chemotherapy. Thereafter, the patientbegan erlotinib, 150 mg daily. A baseline CT scan of the chestdemonstrated innumerable bilateral nodules (FIG. 4B, left panel), whichwere markedly reduced in number and size 4 mo after treatment (FIG. 4B,middle panel). After 14 mo of therapy, the patient's dose of erlotinibwas decreased to 100 mg daily owing to fatigue. At 23 mo of treatmentwith erlotinib, a CT scan demonstrated an enlarging sclerotic lesion inthe thoracic spine. The patient underwent CT-guided biopsy of thislesion (FIG. 5B, left panel), and the erlotinib dose was increased to150 mg daily. After 25 mo of treatment, the disease progressed withinthe lung (FIG. 4B, right panel). Erlotinib was discontinued, and afluoroscopically guided core needle biopsy was performed at a site ofprogressive disease in the lung (FIG. 5B, right panel). In total, thispatient had three tumor specimens available for analysis: the originalresected lung tumor, the biopsy of the enlarging spinal lesion, and thebiopsy of the progressing lung lesion (Table 5).

Patient 3.

This 55-y-old female “never smoker” was treated for nearly 4.5 y withweekly paclitaxel and trastuzumab [17] for adenocarcinoma withbronchioloalveolar carcinoma features involving her left lower lobe,pleura, and mediastinal lymph nodes. Treatment was discontinued owing tofatigue. Subsequently, the patient underwent surgical resection. Becauseof metastatic involvement of multiple mediastinal lymph nodes andclinical features known at that time to be predictive of response togefitinib (female, never smoker, bronchioloalveolar variant histology),she was placed on “adjuvant” gefitinib 1 mo later (FIG. 4C, left panel).This drug was discontinued after three mo when she developed a newleft-sided malignant pleural effusion (FIG. 4C, middle panel). Despitedrainage and systemic chemotherapy, the pleural effusion recurred 4 molater (FIG. 4C, right panel), at which time pleural fluid was collectedfor analysis. In total, this patient had two clinical specimensavailable for analysis: tumor from the surgical resection and pleuralfluid (Table 5).

TABLE 5 Specimens Analyzed in This Study for Mutations in the EGFRTyrosine Kinase Domain (Exons 18 to 24) and KRAS (Exon 2) PercentPathology Specimen Date Tumor Patient Analyzed Obtained Cells EGFR KRAS1 Transbronchial biopsy Day 0 Scant Wild-type Wild-type Progressing lung12 mo >85% L858R + Wild-type lesion T790M Pleural effusion 14 mo >85%L858R + Wild-type T790M 2 Original lung lesion Day 0 >85% del L747-E749;Wild-type A750P Progressing spine 75 mo >85% del L747-E749; Wild-typelesion A750P + T790M Progressing lung 77 mo >85% del L747-E749;Wild-type lesion A750P + T790M 3 Original pleural biopsy Day 0 n/aUnavailable Unavailable Re-resection lung 68 mo >85% del E746-A750Wild-type lesion Pleural effusion 76 mo >50% del E746-A750 + Wild-typeT790M The transbronchial biopsy in patient 1 had scant tumor cells;sequencing analysis revealed only wild-type sequence (see text). Inthree other cases, neither additional EGFR nor KRAS mutations wereidentified (data not shown). Percent tumor cells: defined by assessmentof corresponding histopathological slides. del: deletion; n/a—notapplicable.

Example 5 Patients' Tumors Contain EGFR Tyrosine Kinase Domain MutationsAssociated with Sensitivity to EGFR Tyrosine Kinase Inhibitors

We screened all available tumor samples from the three patientsdescribed in Example 4 for previously described drug-sensitive EGFRmutations, by direct DNA sequencing of exons 19 and 21 [3]. Tumorsamples from patient 1 showed a T change at nucleotide 2573, resultingin the exon 21 L858R amino acid substitution commonly observed indrug-responsive tumors. This mutation was present in the biopsy materialfrom the progressing lung lesion (FIG. 6A, upper panels; Table 5) andfrom cells from the pleural effusion (FIG. 6A, lower panels; Table 5),both of which on cytopathologic examination consisted of a majority oftumor cells (Table 5). Interestingly, comparisons of the tracingssuggest that an increase in copy number of the mutant allele may haveoccurred. Specifically, while the ratio of wild-type (nucleotide T) tomutant (nucleotide G) peaks at position 2573 was approximately 1:1 or1:2 in the lung biopsy specimen (FIG. 6A, upper panels), the pleuralfluid cells demonstrated a dominant mutant G peak (FIG. 6A, lowerpanels). Consistent with this, a single nucleotide polymorphism (SNP)noted at nucleotide 2361 (A or G) demonstrated a corresponding change inthe ratios of A:G, with a 1:1 ratio in the transbronchial biopsy, and anearly 5:1 ratio in the pleural fluid (FIG. 7A). Notably, we did notdetect the 2573 T→G mutation in the original transbronchial biopsyspecimen (Table 5; data not shown). As stated above, this latterspecimen contained scant tumor cells, most likely fewer than needed fordetection of an EGFR mutation by direct sequencing (see [7]).

All three specimens from patient 2, including the original lung tumorand the two metastatic samples from bone and lung, showed an exon 19deletion involving elimination of 11 nucleotides (2238-2248) andinsertion of two nucleotides, G and C (FIG. 6B, all panels; Table 5).These nucleotide changes delete amino acids L747−E749 and change aminoacid 750 from alanine to proline (A750P). A del L747−E749;A750P mutationwas previously reported with different nucleotide changes [2]. In allsamples from patient 2, the wild-type sequence predominated at a ratioof about 3:1 over the mutant sequence.

Both of the available tumor samples from patient 3 contained a deletionof 15 nucleotides (2236-2250) in exon 19 (Table 5; data not shown),resulting in elimination of five amino acids (del E746−A750). Thisspecific deletion has been previously reported [3]. The ratio of mutantto wild-type peaks was approximately 1:1 in both specimens (data notshown).

Collectively, these results demonstrate that tumors from all threepatients contain EGFR mutations associated with sensitivity to thetyrosine kinase inhibitors gefitinib and erlotinib. In addition, thesedata show that within individual patients, metastatic or recurrentlesions to the spine, lung, and pleural fluid contain the samemutations. These latter observations support the idea that relapsing andmetastatic tumor cells within individuals are derived from originalprogenitor clones.

Example 6 A Secondary Missense Mutation in the EGFR Kinase DomainDetected in Lesions that Progressed while on Treatment with EitherGefitinib or Erlotinib

To determine whether additional mutations in the EGFR kinase domain wereassociated with progression of disease in these patients, we performeddirect sequencing of all of the exons (18 through 24) encoding the EGFRcatalytic region in the available tumor specimens.

Analysis of patient 1's pre-gefitinib specimen, which contained scanttumor cells (Table 5; see above), not surprisingly showed only wild-typeEGFR sequence (Table 5; data not shown). However, careful analysis ofthe exon 20 sequence chromatograms in both forward and reversedirections from this patient's lung biopsy specimen obtained afterdisease progression on gefitinib demonstrated an additional small peakat nucleotide 2369, suggesting a C→T mutation (FIG. 7A, upper panels;Table 5). This nucleotide change leads to substitution of methionine forthreonine at position 790 (T790M). The 2369 C→T mutant peak was evenmore prominent in cells from the patient's pleural fluid, which wasobtained after further disease progression on gefitinib (FIG. 7A, lowerpanels; Table 5). The increase in the ratio of mutant to wild-type peaksobtained from analyses of the lung specimen and pleural fluid paralleledthe increase in the ratio of the mutant G peak (leading to the L858Rmutation) to the wild-type T peak at nucleotide 2573 (see above; FIG.6A), as well as the increase in the ratio of the A:G SNP at position2361 (FIG. 7A). Collectively, these findings imply that the exon 20T790M mutation was present on the same allele as the exon 21 L858Rmutation, and that a subclone of cells harboring these mutations emergedduring drug treatment.

In patient 2, the tumor-rich sample obtained prior to treatment witherlotinib did not contain any additional mutations in the exons encodingthe EGFR tyrosine kinase domain (FIG. 7B, upper panels; Table 5). Bycontrast, her progressing bone and lung lesions contained an additionalsmall peak at nucleotide 2369, suggesting the existence of a subclone oftumor cells with the same C→T mutation observed in patient 1 (FIG. 7B,middle and lower panels; Table 5). The relative sizes of the 2369 Tmutant peaks seen in these latter two samples appeared to correlate withthe relative size of the corresponding peaks of the exon 19 deletion(FIG. 6B). Interestingly, the SNP at nucleotide 2361 (A or G) wasdetected in specimens from patient 2 before but not after treatment witherlotinib, suggesting that one EGFR allele underwent amplification ordeletion during the course of treatment (FIG. 6B).

Patient 3 showed results analogous to those of patient 2. A tumor-richpre-treatment specimen did not demonstrate EGFR mutations other than thedel E746−A750 exon 19 deletion; specifically, in exon 20, no secondarychanges were detected (FIG. 7C, upper panels; Table 5). However,analysis of DNA from cells in the pleural effusion that developed aftertreatment with gefitinib showed the C→T mutation at nucleotide 2369 inexon 20 (FIG. 7C, lower panels; Table 5), corresponding to the T790Mmutation described above. There was no dramatic change between the twosamples in the ratio of the A:G SNP at position 2361. The mutant 2369 Tpeak was small, possibly because gefitinib had been discontinued in thispatient for 4 mo at the time pleural fluid tumor cells were collected;thus, there was no selective advantage conferred upon cells bearing theT790M mutation.

To determine whether the 2369 C→T mutation was a previously overlookedEGFR mutation found in NSCLCs, we re-reviewed exon 20 sequence tracingsderived from analysis of 96 fresh-frozen resected tumors [3] and 59paraffin-embedded tumors [7], all of which were removed from patientsprior to treatment with an EGFR tyrosine kinase inhibitor. We did notdetect any evidence of the T790M mutation in these 155 tumors (data notshown). Collectively, our results suggest that the T790M mutation isassociated with lesions that progress while on gefitinib or erlotinib.Moreover, at least in patients 1 and 2, the subclones of tumor cellsbearing this mutation probably emerged between the time of initialtreatment with a tyrosine kinase inhibitor and the appearance of drugresistance.

Additionally, after the initial characterization of the three patientswith the T790M mutation described above in more detail, four otherpatients were found to have the T790M mutation after developingresistance to Iressa or Tarceva out of a total of 13 patients thatinitially responded and then relapsed while on the treatment. Table 6summarizes the results (the table does not include data from the patientthat never responded to treatment and that was later found to haveT790M):

TABLE 6 Time of 1ry 2ry Pt Drug Months Site biopsy Mutation Mutation 1 E19 Spine/lung 26 del T790M 2 G 10 P1 fluid 10 del T790M 3 G 13 Lung 14del T790M 4 G 11 Omentum 12 del T790M 5 G 15 Lung/peric fl 16 del T790M6 G 15 Lung 16 L858R T790M 7 E 16 Lung 22 del none 8 G 11 Lung 13 delnone 9 G 11 P1 fluid/ascites 12 del none 10 G 19 Ascites 23 del none 11G 7 Cervix 8 del none 12 G 12 Ing LN 16 del none 13 G 7 Pleura 9 delnone

In seven patients (case histories not described here) with lungadenocarcinomas who improved but subsequently progressed on therapy witheither gefitinib or erlotinib, we examined DNA from tumor specimensobtained during disease progression. In all seven patients, we foundEGFR mutations associated with drug sensitivity (all exon 19 deletions).However, we did not find any additional mutations in exons 18 to 24 ofEGFR, including the C→T change at position 2369 (data not shown). Theseresults imply that alternative mechanisms of acquired drug resistanceexist.

Example 7 Patients' Progressive Tumors Lack KRAS Mutations

Mutations in exon 2 of KRAS2 occur in about one-fourth of NSCLCs. Suchmutations rarely, if ever, accompany EGFR mutations and are associatedwith primary resistance to gefitinib or erlotinib [7]. To evaluate thepossibility that secondary KRAS mutations confer acquired resistance tothese drugs, we performed mutational profiling of KRAS2 exon 2 fromtumor specimens from patients 1 to 3, as well as the three additionalpatients lacking evidence of the T790M mutation. None of the specimenscontained any changes in KRAS (Table 5; and data not shown), indicatingthat KRAS mutations were not responsible for drug resistance and tumorprogression in these six patients.

Example 8 An Established NSCLC Cell Line Also Contains Both T790M andL858R Mutations

We profiled the EGFR tyrosine kinase domain (exons 18 to 24) and KRASexon 2 in eight established NSCLC lines (Table 7). Surprisingly, onecell line—H1975—contained the same C→T mutation at position 2369 (T790M)as described above (FIG. 7D, lower panel). This cell line had previouslybeen shown by others to contain a 2573 T→G mutation in exon 21 (L858R)[18], which we confirmed (FIG. 7D, upper panel); in addition, H1975 wasreported to be more sensitive to gefitinib inhibition than other lungcancer cell lines bearing wild-type EGFR [18]. Only exons 19 and 21 wereapparently examined in this published study.

TABLE 7 Status of NSCLC Cell Lines Analyzed for EGFR Tyrosine KinaseDomain (Exons 18 to 24) and KRAS (Exon 2) Mutations Cell Line EGFR KRASH1650 del E746-A750 Wild-type H3255 L858R Wild-type H1975 L858R +Wild-type T790M H2030 Wild-type G12C H358 Wild-type G12C H2444 Wild-typeG12V H1734 Wild-type G13C H2347 Wild-type L19F del: deletion. Seemethods for further details.

In our own analysis of H1975 (exons 18 to 24), the mutant 2369 T peakresulting in the T790M amino acid substitution was dominant, suggestingan increase in copy number of the mutant allele in comparison to thewild-type allele. The ratio of mutant to wild-type peaks was similar tothat of the mutant 2573 G (corresponding to the L858R amino acidsubstitution) to wild-type T peaks (FIG. 7D, all panels), implying thatthe T790M and L858R mutations were in the same amplified allele. Tofurther investigate this possibility, we performed RT-PCR to generatecDNAs that spanned exon 20 of EGFR and included sequences from exon 19and 21. PCR products were then cloned, and individual colonies wereanalyzed for EGFR mutations. Sequencing chromatograms of DNA from fourof four clones showed both the 2369 C→T and 2573 T→G mutations,confirming that both mutations were in the same allele (data not shown).

Other NSCLC cell lines carried either EGFR or KRAS mutations, but nonehad both (Table 7). As reported, H3255 contained an L858R mutation [19]and H1650 contained an exon 19 deletion [18]. No other cell linesanalyzed contained additional mutations in the exons encoding the EGFRtyrosine kinase domain.

Example 9 A Novel PCR Restriction Fragment Length Polymorphism AssayIndependently Confirms the Absence or Presence of the T790M Mutation

As stated above, the mutant peaks suggestive of a T790M mutation in exon20 were small in some sequence chromatograms. To eliminate thepossibility that these peaks were due to background “noise,” we soughtto confirm the presence of the 2369 C→T mutation in specific samples, bydeveloping an independent test, based on a fluorescence detection assaythat takes advantage of a PCR restriction fragment length polymorphism(PCR-RFLP) generated by the specific missense mutation. After PCRamplification with exon-20-specific primers spanning nucleotide 2369,wild-type sequence contains specific NlaIII sites, which upon digestionyield a 106-bp product (see Methods; FIG. 3A). Presence of the mutant2369 T nucleotide creates a new NlaIII restriction digest site, yieldinga slightly shorter product (97 bp), readily detected by fluorescentcapillary electrophoresis. This test is about 2-fold more sensitive thandirect sequencing (see Methods; data not shown).

We first used DNA from the H1975 cell line (which contains both T790Mand L858R mutations) to confirm the specificity of the PCR-RFLP assay.As expected, analysis of these cells produced both the 97- and 106-bpfragments. By contrast, analysis of DNA from 112030 (which containswild-type EGFR; Table 7) showed only the 106-bp fragment (FIG. 3A).These data show that this test can readily indicate the absence orpresence of the mutant allele in DNA samples. However, this test wasonly semi-quantitative, as the ratio of the mutant 97-bp product versusthe wild-type 106-bp product varied in independent experiments fromapproximately 1:1 to 2:1.

We next used this PCR-RFLP assay to assess various patient samples forthe presence of the specific 2369 C→T mutation corresponding to theT790M amino acid substitution. DNA from the progressing bone and lunglesions in patient 1 produced both the 97- and 106-bp fragments, but DNAfrom the original lung tumor did not (FIG. 3B). The ratio of mutant towild-type products was higher in the cells from the pleural fluid,consistent with the higher peaks seen on the chromatograms from directsequencing of exon 20 (see FIG. 7A). Likewise, DNA from progressivelesions from patients 2 and 3 yielded both 97- and 106-bp fragments inthe PCR-RFLP assay (FIG. 3B), whereas the pre-treatment specimens didnot produce the 97-bp product. Collectively, these data from anindependent assay confirm that the T790M mutation was present inprogressing lesions from all three patients. We were also unable todetect the T790M mutation in any specimens from the three additionalpatients with acquired resistance that failed to demonstrate secondarymutations in EGFR exons 18 to 24 by direct sequencing (data not shown).

Example 10 Biochemical Properties of EGFR Mutants

To determine how the T790M mutation would affect EGFR proteins alreadycontaining mutations associated with sensitivity to EGFR tyrosine kinaseinhibitors, we introduced the specific mutation into EGFR cDNAs thatencoded the exon 21 and 19 mutations found in patients 1 and 2,respectively. Corresponding proteins ([i] L858R and L858R plus T790M,[ii] del L747−E749;A750P and del L747−E749;A750P plus T790M, and [iii]wild-type EGFR and wild-type EGFR plus T790M) were then produced bytransient transfection with expression vectors in 293T cells, which havevery low levels of endogenous EGFR [3]. Various lysates from cells thatwere serum-starved and pre-treated with gefitinib or erlotinib wereanalyzed by immunoblotting. Amounts of total EGFR (t-EGFR) weredetermined using an anti-EGFR monoclonal antibody, and actin served asan indicator of relative levels of protein per sample. To assess thedrug sensitivity of the various EGFR kinases in surrogate assays, weused a Y1092-phosphate-specific antibody (i.e., phospho-EGFR [p-EGFR])to measure the levels of “autophosphorylated” Tyr-1092 on EGFR inrelation to levels of t-EGFR protein. We also assessed the globalpattern and levels of induced tyrosine phosphorylation of cell proteinsby using a generalized anti-phosphotyrosine reagent (RC-20).

Gefitinib inhibited the activity of wild-type and L858R EGFRsprogressively with increasing concentrations of drug, as demonstrated bya reduction of tyrosine-phosphorylated proteins (FIG. 8A) and a decreasein p-EGFR:t-EGFR ratios (FIG. 8B). By contrast, wild-type and mutantEGFRs containing the T790M mutation did not display a significant changein either phosphotyrosine induction or p-EGFR:t-EGFR ratios (FIGS. 8Aand 8B). Similar results were obtained using erlotinib against wild-typeand del E747−L747;A750P EGFRs in comparison to the corresponding mutantscontaining the T790M mutation (FIG. 8C). These results suggest that theT790M mutation may impair the ability of gefitinib or erlotinib toinhibit EGFR tyrosine kinase activity, even in EGFR mutants (i.e., L858Ror an exon 19 deletion) that are clinically associated with drugsensitivity.

Example 11 Resistance of a NSCLC Cell Line Harboring Both T790M andL858R Mutations to Gefitinib or Erlotinib

To further explore the functional consequences of the T790M mutation, wedetermined the sensitivity of various NSCLC cells lines grown in thepresence of either gefitinib or erlotinib, using an assay based uponCalcein AM. Uptake and retention of this fluorogenic esterase substrateby vehicle-versus drug-treated live cells allows for a comparison ofrelative cell viability among cell lines [20]. The H3255 cell line,which harbors the L858R mutation and no other EGFR TK domain mutations(Table 7), was sensitive to treatment with gefitinib, with an IC₅₀ ofabout 0.01 μmol (FIG. 9). By contrast, the H1975 cell line, whichcontains both L858R and T790M mutations (Table 7), was approximately100-fold less sensitive to drug, with an IC₅₀ of about 1 mol (FIG. 9).In fact, the sensitivity of H1975 cells was more similar to that ofH2030, which contains wild-type EGFR (exons 18 to 24) and mutant KRAS(FIG. 9). Very similar results were obtained with erlotinib (FIG. 10).

Although the present invention may have been disclosed and illustratedherein by reference to exemplary embodiments thereof, all equivalentembodiments, including alterations, additions and omissions, areencompassed within the spirit and scope of the invention as disclosed inthe specification and the claims.

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We claim:
 1. A method for predicting development of acquired resistanceto the therapeutic effects of an epidermal growth factor (EGFR) tyrosinekinase inhibitor in a patient that is suffering from a cancer, whereinthe method comprises the steps of: (a) obtaining a sample from thepatient, wherein the cancer harbors a somatic gain-of-function mutationin the tyrosine kinase domain of EGFR that enhances the sensitivity ofthe cancer to the tyrosine kinase inhibitor, (b) testing the sample todetermine whether the gene encoding EGFR is present in a mutant formthat encodes a T790M mutant of EGFR in addition to the somatic gain offunction mutation, and (c) identifying the cancer as one that has becomeor will become resistant to the EGFR tyrosine kinase inhibitor if theT790M mutant form is present or as having continuing sensitivity to theEGFR tyrosine kinase inhibitor if the T790M mutant form is absent,wherein the testing of the sample in step (b) is performed by a methodselected from the group consisting of: (i) performing PCR amplificationusing a pair of primers that flank the region encoding amino acid 790 ofEGFR to form amplicons that include the bases encoding amino acid 790 ofEGFR, and evaluating the amplicons to determine if a mutation is presentthat would result in a T790M mutation in EGFR; (ii) performing PCRamplification using a pair of primers, and evaluating the amplicons todetermine if a mutation is present that would result in a T790M mutationin EGFR, and wherein one of the primers used in the PCR amplificationstep binds at a position that includes the bases encoding a T790Mmutation in EGFR; and (iii) probing the sample with a probeoligonucleotide, wherein the probe binds preferentially to a 2369C→Tmutant or a wild type EGFR sequence, and detecting binding of the probe.2. The method of claim 1, wherein the patient is treated with the EGFRtyrosine kinase inhibitor prior to obtaining the sample from thepatient.
 3. The method of claim 2, wherein the patient is responsive tothe EGFR tyrosine kinase inhibitor when it is first administered.
 4. Themethod of claim 1, wherein the somatic gain-of-function mutation isL858R.
 5. The method of claim 1, wherein the somatic gain-of-functionmutation is a deletion of the amino acid sequence Leu-Arg-Glu-Ala fromexon 19 of EGFR.
 6. The method of claim 1, wherein the EGFR tyrosinekinase inhibitor is gefitinib or erlotinib.
 7. The method of claim 6,wherein the patient is treated with the EGFR tyrosine kinase inhibitorprior to obtaining the sample from the patient.
 8. The method of claim6, wherein the somatic gain-of-function mutation is L858R.
 9. The methodof claim 6, wherein the somatic gain-of-function mutation is a deletionof the amino acid sequence Leu-Arg-Glu-Ala from exon 19 of EGFR.
 10. Themethod of claim 6, wherein the somatic gain-of-function mutation is adeletion from exon 19 of EGFR.
 11. The method of claim 1, wherein thecancer is non-small cell lung cancer.
 12. The method of claim 7, whereinthe patient is responsive to the EGFR tyrosine kinase inhibitor when itis first administered.
 13. The method of claim 11, wherein the EGFRtyrosine kinase inhibitor is gefitinib or erlotinib.
 14. The method ofclaim 1, wherein the step of testing the sample comprises the steps of:performing PCR amplification using a pair of primers that flank theregion encoding amino acid 790 of EGFR to form amplicons that includethe bases encoding amino acid 790 of EGFR, and evaluating the ampliconsto determine if a mutation is present that would result in a T790Mmutation in EGFR.
 15. The method of claim 14, wherein the pair ofprimers includes at least one primer selected from among Seq ID Nos 4-7.16. The method of claim 14, wherein the testing step further comprisesthe step of exposing the amplicons to a cleaving means, said cleavingmeans cleaves one but both not both of the wild type and mutantamplicons.
 17. The method of claim 1, wherein the step of testing thesample comprises the steps of: performing PCR amplification using a pairof primers, and evaluating the amplicons to determine if a mutation ispresent that would result in a T790M mutation in EGFR, and wherein oneof the primers used in the PCR amplification step binds at a positionthat includes the bases encoding a T790M mutation in EGFR.
 18. Themethod of claim 17, wherein the primer that binds at a positionincluding the bases encoding a T790M mutation in EGFR has a sequencesuch that the wild type genes and mutant genes are differentiallyamplified.
 19. The method of claim 17, wherein the primer that binds ata position including the bases encoding a T790M mutation in EGFR isselected from among Seq ID Nos. 12-15.
 20. The method of claim 1,wherein the step of testing comprises the steps of probing the samplewith a probe oligonucleotide, wherein the probe binds preferentially toa 2369→T mutant or a wild type EGFR sequence, and detecting binding ofthe probe.
 21. The method of claim 20, wherein the probe oligonucleotideis immobilized.
 22. The method of claim 1, wherein the somaticgain-of-function mutation is a deletion from exon 19 of EGFR.
 23. Themethod of claim 22, wherein the EGFR tyrosine kinase inhibitor isgefitinib or erlotinib.
 24. A method for predicting development ofacquired resistance to the therapeutic effects of a means for inhibitingepidermal growth factor (EGFR) tyrosine kinase in a patient that issuffering from a cancer, wherein the method comprises the steps of: (a)obtaining a sample from the patient, wherein the cancer harbors asomatic gain-of-function mutation in the tyrosine kinase domain of EGFRthat enhances the sensitivity of the cancer to the means for inhibitingEGFR tyrosine kinase, (b) testing the sample to determine whether thegene encoding EGFR is present in a mutant form that encodes a T790Mmutant of EGFR in addition to the somatic gain of function mutation, and(c) identifying the cancer as one that has become or will becomeresistant to the means for inhibiting EGFR tyrosine kinase if the T790Mmutant form is present or as having continuing sensitivity to the meansfor inhibiting EGFR tyrosine kinase if the T790M mutant form is absent,wherein the testing of the sample in step (b) is performed by a methodselected from the group consisting of: (i) performing PCR amplificationusing a pair of primers that flank the region encoding amino acid 790 ofEGFR to form amplicons that include the bases encoding amino acid 790 ofEGFR, and evaluating the amplicons to determine if a mutation is presentthat would result in a T790M mutation in EGFR; (ii) performing PCRamplification using a pair of primers, and evaluating the amplicons todetermine if a mutation is present that would result in a T790M mutationin EGFR, and wherein one of the primers used in the PCR amplificationstep binds at a position that includes the bases encoding a T790Mmutation in EGFR; and (iii) probing the sample with a probeoligonucleotide, wherein the probe binds preferentially to a 2369C→Tmutant or a wild type EGFR sequence, and detecting binding of the probe.